\u062f PRF \u062d\u062c\u0631\u0648 \u0644\u0627\u06cc\u0646\u0648\u0646\u0647 \u063a\u0648\u0693\u0648\u0644<\/strong><\/h4>\n\n- \u062d\u062c\u0631\u06d0 \u067e\u0647 \u0686\u067c\u06a9\u06cd \u0633\u0631\u0647 \u067e\u0647 37\u02daC \u062f \u0627\u0648\u0628\u0648 \u062d\u0645\u0627\u0645 \u06a9\u06d0 \u0648\u063a\u0648\u0631\u0681\u0648\u0626.<\/li>\n
- \u062f \u0634\u06cc\u0634\u06d0 \u0628\u0647\u0631 \u062f 70% \u0627\u06cc\u062a\u0627\u0646\u0648\u0644 \u0633\u0631\u0647 \u067e\u0627\u06a9 \u06a9\u0693\u0626.<\/li>\n
- \u067e\u0693\u0633\u06cc\u062f\u0644\u064a \u062d\u062c\u0631\u06d0 \u062f T25 \u0641\u0644\u0627\u0633\u06a9 \u062a\u0647 \u0627\u0646\u062a\u0642\u0627\u0644 \u06a9\u0693\u0626 \u0686\u06d0 \u062f \u0648\u062f\u06d0 \u0645\u06cc\u0689\u06cc\u0627 5 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631\u0647 \u0644\u0631\u064a.<\/li>\n
- \u0641\u0644\u0627\u0633\u06a9 \u062f \u0634\u067e\u06d0 \u067e\u0647 37\u02daC \u0627\u0646\u06a9\u06cc\u0648\u0628\u06cc\u067c\u0631 \u06a9\u06d0 \u0681\u0627\u06cc \u067e\u0647 \u0681\u0627\u06cc \u06a9\u0693\u0626.<\/li>\n
- \u0628\u0644\u0647 \u0648\u0631\u0681\u060c \u062f \u0645\u0627\u06cc\u06a9\u0631\u0648\u0633\u06a9\u0648\u067e \u0644\u0627\u0646\u062f\u06d0 \u0648\u06ab\u0648\u0631\u0626 \u062a\u0631\u0685\u0648 \u0689\u0627\u0689 \u062a\u0631\u0644\u0627\u0633\u0647 \u06a9\u0693\u0626 \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u062a\u0693\u0644 \u0634\u0648\u064a \u062f\u064a.<\/li>\n
- \u0631\u0633\u0646\u06cd \u0644\u0631\u06d0 \u06a9\u0693\u0626 \u0627\u0648 \u062f \u062a\u0627\u0632\u0647 \u0648\u062f\u06d0 \u0645\u06cc\u0689\u06cc\u0627 \u0633\u0631\u0647 \u0681\u0627\u06cc \u067e\u0647 \u0681\u0627\u06cc \u06a9\u0693\u0626.<\/li>\n<\/ol>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n\n\n\n\n\n\n
\u062f PRF \u0633\u06cc\u0644 \u0644\u06cc\u0646\u0648\u0646\u0648 \u0641\u0631\u0639\u064a \u06a9\u0644\u062a\u0648\u0631 \u06a9\u0648\u0644<\/strong><\/h4>\n\n- \u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u062a\u0642\u0633\u06cc\u0645 \u0634\u064a \u06a9\u0644\u0647 \u0686\u06d0 \u06cc\u0648 \u0681\u0627\u06cc \u0634\u064a.<\/li>\n
- \u062f \u062d\u062c\u0631\u0648 \u062f \u0648\u06cc\u0634\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 (\u062f \u0648\u0631\u06a9\u0693\u0644 \u0634\u0648\u06cc\u0648 \u062d\u062c\u0645\u0648\u0646\u0648 \u0685\u062e\u0647 \u0641\u0631\u0636 \u06a9\u06cc\u0696\u064a \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u067e\u0647 T25 \u06a9\u06d0 \u062f\u064a):
a) \u062f \u062c\u0631\u0627\u062b\u06cc\u0645 \u0636\u062f \u062a\u062e\u0646\u06cc\u06a9 \u067e\u0647 \u06a9\u0627\u0631\u0648\u0644\u0648 \u0633\u0631\u0647 \u060c \u0645\u06cc\u0689\u06cc\u0627 \u0644\u0631\u06d0 \u06a9\u0693\u0626 \u0627\u0648 \u0641\u0644\u0627\u0633\u06a9 \u062f 2-3 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u062c\u0631\u0627\u062b\u06cc\u0645\u064a HBSS \u0633\u0631\u0647 \u0648\u06cc\u0646\u0681\u0626. HBSS \u0644\u0631\u06d0 \u0627\u0648 \u0631\u062f \u06a9\u0693\u0626.
b) 1 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u067c\u0631\u06cc\u067e\u0633\u06cc\u0646 \u0627\u0636\u0627\u0641\u0647 \u06a9\u0693\u0626 \u0627\u0648 \u062f 2-3 \u062f\u0642\u06cc\u0642\u0648 \u0644\u067e\u0627\u0631\u0647 \u06cc\u06d0 \u0648\u06cc\u0646\u0681\u0626. \u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u062f \u0627\u0644\u062a\u0647\u0627\u0628 \u0645\u0627\u06cc\u06a9\u0631\u0648\u0633\u06a9\u0648\u067e \u0644\u0627\u0646\u062f\u06d0 \u0648\u0685\u06cc\u0693\u0644 \u0634\u064a \u062a\u0631\u0685\u0648 \u0645\u0639\u0644\u0648\u0645\u0647 \u06a9\u0693\u064a \u0686\u06d0 \u0627\u06cc\u0627 \u062f\u0648\u06cc \u06ab\u0631\u062f\u06cc\u062f\u0644\u060c \u067e\u0648\u0631\u062a\u0647 \u06a9\u0648\u0644 \u0627\u0648 \u062a\u06cc\u0631\u06cc\u062f\u0644 \u067e\u06cc\u0644 \u06a9\u0693\u064a \u062f\u064a. \u06a9\u0647 \u062d\u062c\u0631\u06d0 \u062f 3 \u062f\u0642\u06cc\u0642\u0648 \u0648\u0631\u0648\u0633\u062a\u0647 \u062c\u0644\u0627 \u0646\u0647 \u0634\u064a\u060c \u062a\u0627\u0633\u0648 \u06a9\u0648\u0644\u06cc \u0634\u0626 \u062f 1-2 \u062f\u0642\u06cc\u0642\u0648 \u0644\u067e\u0627\u0631\u0647 \u0628\u0644 \u0681\u0627\u06cc \u0648\u0646\u06cc\u0633\u0626.
c) \u06a9\u06cc\u067e \u067c\u06cc\u0646\u06ab \u06a9\u0693\u0626 \u0627\u0648 \u067e\u0647 \u0646\u0631\u0645\u06cd \u0633\u0631\u0647 \u0641\u0644\u0627\u0633\u06a9 \u0644\u0647 \u06cc\u0648\u06d0 \u062e\u0648\u0627 \u0685\u062e\u0647 \u0628\u0644\u06d0 \u062e\u0648\u0627 \u062a\u0647 \u0648\u062e\u0648\u0631\u0626 \u062a\u0631\u0685\u0648 \u067c\u0648\u0644\u06d0 \u062d\u062c\u0631\u06d0 \u0644\u0647 \u0645\u06cc\u0646\u0681\u0647 \u0648\u06cc\u0633\u064a. \u0641\u0644\u0627\u0633\u06a9 \u0645\u0645\u06a9\u0646 \u062f \u0627\u0693\u062a\u06cc\u0627 \u067e\u0647 \u0635\u0648\u0631\u062a \u06a9\u06d0 \u062f \u062d\u062c\u0631\u0648 \u062f \u062e\u0644\u0627\u0635\u0648\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u067e\u0647 \u06cc\u0648 \u0627\u0693\u062e \u06a9\u06d0 \u067e\u0647 \u0646\u0631\u0645\u06cd \u0633\u0631\u0647 \u067c\u067e \u0634\u064a.
d) \u06a9\u0644\u0647 \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u062f \u067c\u0631\u06cc\u067e\u0633\u06cc\u0646 \u063a\u06cc\u0631 \u0641\u0639\u0627\u0644\u0648\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u062a\u06cc\u0631\u06cc\u0696\u064a \u067e\u0647 \u0641\u0644\u0627\u0633\u06a9 \u06a9\u06d0 4 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u0646\u0648\u064a \u0645\u06cc\u0689\u06cc\u0627 \u0627\u0636\u0627\u0641\u0647 \u06a9\u0693\u0626. \u062f \u0641\u0644\u0627\u0633\u06a9 \u0627\u0693\u062e\u0648\u0646\u0647 \u0685\u0648 \u0681\u0644\u0647 \u062f \u0646\u0648\u064a \u0645\u06cc\u0689\u06cc\u0627 \u0633\u0631\u0647 \u0645\u06cc\u0646\u0681\u0644 \u06a9\u0693\u0626 \u062a\u0631\u0685\u0648 \u067c\u0648\u0644 \u062d\u062c\u0631\u06d0 \u0644\u0647 \u067e\u0644\u0627\u0633\u062a\u06cc\u06a9 \u0685\u062e\u0647 \u067e\u0627\u06a9 \u0627\u0648 \u0645\u062d\u0644\u0648\u0644 \u062a\u0647 \u0648\u0645\u06cc\u0646\u0681\u0626. \u067c\u0648\u0644 \u0645\u0627\u06cc\u0639 \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u0644\u0631\u064a \u0644\u0631\u06d0 \u06a9\u0693\u0626 \u0627\u0648 \u062f 15 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u0645\u062e\u0631\u0648\u0637 (\u06cc\u0627 50 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631) \u062a\u0647 \u06cc\u06d0 \u0627\u0646\u062a\u0642\u0627\u0644 \u06a9\u0693\u0626 \u06a9\u0647 \u062a\u0627\u0633\u0648 3 \u06cc\u0627 \u0689\u06cc\u0631 \u0641\u0644\u0627\u0633\u06a9\u0648\u0646\u0647 \u0631\u0627\u067c\u0648\u0644 \u06a9\u0693\u0626.
e) \u062f \u062c\u0631\u0627\u062b\u06cc\u0645\u064a \u062a\u062e\u0646\u06cc\u06a9 \u067e\u0647 \u06a9\u0627\u0631\u0648\u0644\u0648 \u0633\u0631\u0647\u060c \u062f \u0647\u06cc\u0645\u0648\u0633\u0627\u06cc\u067c\u0648\u0645\u06cc\u067c\u0631 \u062f \u0634\u0645\u06cc\u0631\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u0627\u0644\u06cc\u06a9\u0648\u067c \u0644\u0631\u06d0 \u06a9\u0693\u0626.
f) \u067e\u062f\u0627\u0633\u06d0 \u062d\u0627\u0644 \u06a9\u06d0 \u0686\u06d0 \u062a\u0627\u0633\u0648 \u062d\u062c\u0631\u06d0 \u0634\u0645\u06cc\u0631\u0626\u060c \u067e\u0627\u062a\u06d0 \u0645\u062d\u0644\u0648\u0644 \u0628\u0627\u06cc\u062f \u067e\u0647 \u06a9\u0644\u06cc\u0646\u06cc\u06a9\u064a \u0633\u0646\u067c\u0631\u0641\u06cc\u0648\u062c \u06a9\u06d0 \u062f 5 \u062f\u0642\u06cc\u0642\u0648 \u0644\u067e\u0627\u0631\u0647 \u067e\u0647 1000 rpm \u06a9\u06d0 \u0648\u0627\u0686\u0648\u0644 \u0634\u064a.<\/li>\n - \u0633\u062a\u0627\u0633\u0648 \u062f \u062d\u062c\u0631\u0648 \u0634\u0645\u06cc\u0631\u0647 \u0645\u062d\u0627\u0633\u0628\u0647 \u06a9\u0648\u0644\u0648 \u0648\u0631\u0648\u0633\u062a\u0647\u060c \u067e\u0647 2.5 x 10 \u06a9\u06d0 \u062d\u062c\u0631\u06d0 \u067e\u0644\u06cc\u067c \u06a9\u0693\u06265<\/sup>\u00a0<\/span>\u062d\u062c\u0631\u06d0 \u067e\u0647 \u0647\u0631 T25 \u0641\u0644\u067c\u0631 \u067c\u0627\u067e \u0641\u0644\u0627\u0633\u06a9 \u06a9\u06d0.<\/li>\n
- \u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u067e\u0647 \u0647\u0631\u0648 2-3 \u0648\u0631\u0681\u0648 \u06a9\u06d0 \u062f \u062a\u0627\u0632\u0647 \u0648\u062f\u06d0 \u0645\u0646\u0681\u0646\u064a \u0633\u0631\u0647 \u062a\u063a\u0630\u06cc\u0647 \u0634\u064a.<\/li>\n<\/ol>\n
\u06cc\u062e \u06a9\u0648\u0644:
<\/u><\/strong><\/h4>\n\u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u0644\u0647 5 x 10 \u0685\u062e\u0647 \u06a9\u0645 \u0646\u0647 \u0648\u064a \u0645\u0646\u062c\u0645\u062f \u0634\u064a5<\/sup>\u00a0<\/span>\u062d\u062c\u0631\u06d0\/ml\/cryovial \u062f \u0648\u062f\u06d0 \u0645\u06cc\u0689\u06cc\u0627 \u06a9\u06d0 \u0686\u06d0 10% DMSO \u0627\u0648 30% FBS \u0644\u0631\u064a \u0627\u0648 \u0648\u0631\u0648\u0633\u062a\u0647 \u062f \u0634\u067e\u06d0 \u067e\u0647 -80\u02daC \u06a9\u06d0 \u062f isopropanol \u067e\u0647 \u06cc\u062e\u0686\u0627\u0644 \u06a9\u06d0 \u0681\u0627\u06cc \u067e\u0647 \u0681\u0627\u06cc \u0634\u0648\u064a. \u0628\u0644\u0647 \u0648\u0631\u0681 \u0645\u0627\u06cc\u0639 \u0646\u0627\u06cc\u067c\u0631\u0648\u062c\u0646 \u062a\u0647 \u0627\u0646\u062a\u0642\u0627\u0644 \u06a9\u0693\u0626.<\/p>\n\u062f \u0641\u0631\u0639\u064a \u06a9\u0644\u062a\u0648\u0631 \u06a9\u0648\u0644\u0648 \u0627\u0648 \u0645\u0646\u062c\u0645\u062f \u0641\u06cc\u0628\u0631\u0648\u0628\u0644\u0627\u0633\u067c\u0648\u0646\u0648 \u067e\u0631\u0648\u062a\u0648\u06a9\u0648\u0644<\/a><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_tablet=\u201d|35px||35px||\u0631\u06cc\u069a\u062a\u06cc\u0627\u201d custom_padding_phone=\u201d\u201dborder_width_top=\u201d10px\u201dborder_color_top=\u201d#8fd2ed\u201d \u0628\u0646\u062f =\u201d\u0628\u0646\u062f\u201d Global_colors_info=\u201d{}\u201d][et_pb_column_info="{}"][et_pb_column_info="_inner_type_4_Seave" _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d Global_colors_info="{}" custom_padding__hover="|||"][et_pb_text _builder_version="4.16\u2033 global_colors_info="{}"]<\/p>\n
\u062f \u0646\u0648\u0631\u0648 \u0645\u0639\u0644\u0648\u0645\u0627\u062a\u0648 \u0644\u067e\u0627\u0631\u0647 \u0645\u0647\u0631\u0628\u0627\u0646\u064a \u0648\u06a9\u0693\u0626 \u0627\u0693\u06cc\u06a9\u0647 \u0648\u0646\u06cc\u0633\u0626:<\/strong><\/h4>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\u201d on|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d|35px|0|0px|\u063a\u0644\u0637 ="1_2\u2033 \u062e\u0648\u0646\u062f\u064a \u0634\u0648\u06cc_\u062e\u0627\u0635\u06cc\u062a_\u06a9\u0627\u0644\u0645_\u0689\u0648\u0644 =\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|20px||\u201d custom_padding_tablet="||20px" custom_padding_phone="" custom_padding_last_edited="on|desktop" global_colors_info="{}" custom_padding__hover="|||"][et_pb_text _builder_version="4.19.5\u2033 \u067e\u0647 \u0646\u0693\u06cc\u0648\u0627\u0644 \u06a9\u06d0"}_colors"}<\/p>\n
\u0644\u06cc\u0633\u0644\u064a \u0628\u06cc \u06ab\u0648\u0631\u0689\u0646\u060c MD\u060c PhD<\/h6>\n
\u062f \u0628\u0631\u0627\u0648\u0646 \u067e\u0648\u0647\u0646\u062a\u0648\u0646 \u062f \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u062f \u0685\u06cc\u0693\u0646\u06d0 \u067e\u0631\u0648\u0641\u06cc\u0633\u0648\u0631 \u0648\u0627\u0631\u0646 \u0627\u0644\u067e\u0631\u067c \u0637\u0628\u064a \u069a\u0648\u0648\u0646\u0681\u064a \u0627\u0648 \u062f \u067e\u06cc\u0689\u06cc\u0627\u067c\u0631\u06cc\u06a9\u0633 \u0689\u06cc\u067e\u0627\u0631\u067c\u0645\u0646\u067c \u060c \u062f \u0647\u0627\u0633\u0628\u0631\u0648 \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u0631\u0648\u063a\u062a\u0648\u0646 \u060c \u067e\u0631\u0648\u0648\u06cc\u0689\u0646\u0633 \u060c \u062f \u0627\u0646\u0633\u062a\u06cc\u0632\u06cc\u0627 RI \u0685\u0627\u0646\u06ab\u0647 \u060c \u062f \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u0631\u0648\u063a\u062a\u0648\u0646 \u0628\u0648\u0633\u067c\u0646 \u0627\u0648 \u0647\u0627\u0631\u0648\u0631\u0689 \u0645\u06cc\u0689\u06cc\u06a9\u0644 \u069a\u0648\u0648\u0646\u0681\u06cc \u060c \u0628\u0648\u0633\u067c\u0646 \u060c \u062f MA \u0637\u0628\u064a \u0631\u06cc\u06cc\u0633 \u060c \u062f \u067e\u0631\u0648\u062c\u06cc\u0631\u06cc\u0627 \u0631\u06cc\u0633\u0631\u0686 \u0641\u0627\u0648\u0646\u0689\u06cc\u0634\u0646<\/p>\n
\u062a\u0644\u06cc\u0641\u0648\u0646: 978-535-2594
\u0641\u06a9\u0633: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n] custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|phone\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_last_edited=\u201doff|desktop\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033]<\/p>\n
\u0648\u06cc\u0646\u0689\u064a \u0646\u0648\u0631\u06cc\u0633<\/span><\/h6>\n\u062f PRF \u0633\u06cc\u0644 \u0627\u0648 \u0646\u0633\u062c \u0628\u0627\u0646\u06a9
\u062a\u0644\u06cc\u0641\u0648\u0646: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
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[\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info="{}" custom_padding__hover="|||"][et_pb_image src="https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2024\/08\/2024-strip-footer-strip-copy.png "\u0633\u0631\u0644\u06cc\u06a9_\u0645\u062a\u0646 ="2024 \u067e\u067c\u06d0 \u0641\u0648\u067c\u0631 \u067e\u067c\u06d0 \u06a9\u0627\u067e\u064a" _builder_version="4.27.0\u2033 _module_preset="default" custom_margin="35px||||false|false" global_colors_info="{}"][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_ction]<\/p>","protected":false},"excerpt":{"rendered":"
[et_pb_section fb_built=”1″ fullwidth=”on” disabled_on=”off|off|off” _builder_version=”4.16″ border_width_bottom=”55px” border_color_bottom=”#29327a” locked=”off” global_colors_info=”{}”][et_pb_fullwidth_header _builder_version=”4.16″ title_font=”||||||||” title_font_size=”55″ background_color=”#29327a” background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg” background_position=”center_left” custom_padding=”9vw||9vw||true” custom_padding_tablet=”” custom_padding_phone=”|56px||” custom_padding_last_edited=”on|desktop” title_font_size_tablet=”45px” title_font_size_phone=”40px” title_font_size_last_edited=”on|phone” z_index_tablet=”500″ custom_css_main_element=”background-position: center 18% !important;” global_colors_info=”{}”] Fibroblast Cell Culture Protocols [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” […]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Fibroblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n
PROTOCOL FOR SUBCULTURING AND FREEZING FIBROBLASTS\u00a0<\/strong><\/p>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=\"1\/2\"][vc_column_text]Growth Media<\/u><\/strong>\r\n\r\nDMEM - ThermoFisher #11960-044 (high glucose without L-glutamine)\r\n\r\n15% FBS Fetal Bovine Serum \u2013 ThermoFisher #10437-028\r\n\r\n1% (1X) Penicillin-Streptomycin - ThermoFisher #15140-122\r\n\r\n1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061[\/vc_column_text][\/vc_column][vc_column width=\"1\/2\"][vc_column_text]Trypsin<\/u><\/strong>\r\n\r\nTrypsin EDTA C 0.25% (ThermoFisher #25200-056)\r\n\r\nHank\u2019s Balanced Salt Solution\r\n<\/u><\/strong>HBSS- (ThermoFisher #14170 (1X) (-) calcium chloride, (-) magnesium chloride, (-) magnesium sulfate\r\n\r\nDMSO for Freezing\r\n<\/u><\/strong>Cell Culture Grade DMSO - Sigma #D2438[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Thawing PRF cell lines<\/u><\/strong>\r\n\r\n \t- Thaw cells rapidly in a 37\u02daC water bath.<\/li>\r\n \t
- Wipe the outside of the vial with 70% ethanol.<\/li>\r\n \t
- Transfer the thawed cells to a T25 flask containing 5 ml of growth media.<\/li>\r\n \t
- Place flask in 37\u02daC incubator overnight.<\/li>\r\n \t
- The following day, observe under the microscope to ensure cells have attached.<\/li>\r\n \t
- Remove media and replace with fresh growth media.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Subculturing PRF cell lines<\/u><\/strong>\r\n
\r\n \t- Cells should be split when confluent.<\/li>\r\n \t
- To split cells (volumes given are assuming cells are in a T25):\r\na)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.\r\nb)\u00a0 Add 1 ml trypsin and incubate for 2-3 minutes. Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float.\u00a0 If cells are not detached after 3 minutes, you may incubate another 1-2 minutes.\r\nc)\u00a0 Tighten cap and gently tip flask from side to side to dislodge all cells. Flask may be tapped lightly on side to loosen cells if needed.\r\nd)\u00a0 Add 4 ml of new media to flask as soon as cells are floating to inactivate the trypsin. Rinse down sides of flask several times with the new media to wash all cells off the plastic and into the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).\r\ne)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.\r\nf)\u00a0 While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpm.<\/li>\r\n \t
- After calculating your cell count, plate cells at 2.5 x 105<\/sup> cells per T25 filter top flask.<\/li>\r\n \t
- Cells should be fed every 2-3 days with fresh growth medium.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Freezing:\r\n<\/u><\/strong>Cells should be frozen at no less than 5 x 105<\/sup> cells\/ml\/cryovial in growth media containing 10% DMSO and 30% FBS and subsequently placed in an isopropanol freezing chamber at -80\u02daC overnight.\u00a0 Transfer to the liquid nitrogen the next day.\r\n\r\n
![\"\"](\"https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2017\/04\/iconPDF.gif\")
\u00a0Protocol for Subculturing and Freezing Fibroblasts<\/a>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA,\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n
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c) \u06a9\u06cc\u067e \u067c\u06cc\u0646\u06ab \u06a9\u0693\u0626 \u0627\u0648 \u067e\u0647 \u0646\u0631\u0645\u06cd \u0633\u0631\u0647 \u0641\u0644\u0627\u0633\u06a9 \u0644\u0647 \u06cc\u0648\u06d0 \u062e\u0648\u0627 \u0685\u062e\u0647 \u0628\u0644\u06d0 \u062e\u0648\u0627 \u062a\u0647 \u0648\u062e\u0648\u0631\u0626 \u062a\u0631\u0685\u0648 \u067c\u0648\u0644\u06d0 \u062d\u062c\u0631\u06d0 \u0644\u0647 \u0645\u06cc\u0646\u0681\u0647 \u0648\u06cc\u0633\u064a. \u0641\u0644\u0627\u0633\u06a9 \u0645\u0645\u06a9\u0646 \u062f \u0627\u0693\u062a\u06cc\u0627 \u067e\u0647 \u0635\u0648\u0631\u062a \u06a9\u06d0 \u062f \u062d\u062c\u0631\u0648 \u062f \u062e\u0644\u0627\u0635\u0648\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u067e\u0647 \u06cc\u0648 \u0627\u0693\u062e \u06a9\u06d0 \u067e\u0647 \u0646\u0631\u0645\u06cd \u0633\u0631\u0647 \u067c\u067e \u0634\u064a.
d) \u06a9\u0644\u0647 \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u062f \u067c\u0631\u06cc\u067e\u0633\u06cc\u0646 \u063a\u06cc\u0631 \u0641\u0639\u0627\u0644\u0648\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u062a\u06cc\u0631\u06cc\u0696\u064a \u067e\u0647 \u0641\u0644\u0627\u0633\u06a9 \u06a9\u06d0 4 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u0646\u0648\u064a \u0645\u06cc\u0689\u06cc\u0627 \u0627\u0636\u0627\u0641\u0647 \u06a9\u0693\u0626. \u062f \u0641\u0644\u0627\u0633\u06a9 \u0627\u0693\u062e\u0648\u0646\u0647 \u0685\u0648 \u0681\u0644\u0647 \u062f \u0646\u0648\u064a \u0645\u06cc\u0689\u06cc\u0627 \u0633\u0631\u0647 \u0645\u06cc\u0646\u0681\u0644 \u06a9\u0693\u0626 \u062a\u0631\u0685\u0648 \u067c\u0648\u0644 \u062d\u062c\u0631\u06d0 \u0644\u0647 \u067e\u0644\u0627\u0633\u062a\u06cc\u06a9 \u0685\u062e\u0647 \u067e\u0627\u06a9 \u0627\u0648 \u0645\u062d\u0644\u0648\u0644 \u062a\u0647 \u0648\u0645\u06cc\u0646\u0681\u0626. \u067c\u0648\u0644 \u0645\u0627\u06cc\u0639 \u0686\u06d0 \u062d\u062c\u0631\u06d0 \u0644\u0631\u064a \u0644\u0631\u06d0 \u06a9\u0693\u0626 \u0627\u0648 \u062f 15 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631 \u0645\u062e\u0631\u0648\u0637 (\u06cc\u0627 50 \u0645\u0644\u06cc \u0644\u06cc\u062a\u0631) \u062a\u0647 \u06cc\u06d0 \u0627\u0646\u062a\u0642\u0627\u0644 \u06a9\u0693\u0626 \u06a9\u0647 \u062a\u0627\u0633\u0648 3 \u06cc\u0627 \u0689\u06cc\u0631 \u0641\u0644\u0627\u0633\u06a9\u0648\u0646\u0647 \u0631\u0627\u067c\u0648\u0644 \u06a9\u0693\u0626.
e) \u062f \u062c\u0631\u0627\u062b\u06cc\u0645\u064a \u062a\u062e\u0646\u06cc\u06a9 \u067e\u0647 \u06a9\u0627\u0631\u0648\u0644\u0648 \u0633\u0631\u0647\u060c \u062f \u0647\u06cc\u0645\u0648\u0633\u0627\u06cc\u067c\u0648\u0645\u06cc\u067c\u0631 \u062f \u0634\u0645\u06cc\u0631\u0644\u0648 \u0644\u067e\u0627\u0631\u0647 \u0627\u0644\u06cc\u06a9\u0648\u067c \u0644\u0631\u06d0 \u06a9\u0693\u0626.
f) \u067e\u062f\u0627\u0633\u06d0 \u062d\u0627\u0644 \u06a9\u06d0 \u0686\u06d0 \u062a\u0627\u0633\u0648 \u062d\u062c\u0631\u06d0 \u0634\u0645\u06cc\u0631\u0626\u060c \u067e\u0627\u062a\u06d0 \u0645\u062d\u0644\u0648\u0644 \u0628\u0627\u06cc\u062f \u067e\u0647 \u06a9\u0644\u06cc\u0646\u06cc\u06a9\u064a \u0633\u0646\u067c\u0631\u0641\u06cc\u0648\u062c \u06a9\u06d0 \u062f 5 \u062f\u0642\u06cc\u0642\u0648 \u0644\u067e\u0627\u0631\u0647 \u067e\u0647 1000 rpm \u06a9\u06d0 \u0648\u0627\u0686\u0648\u0644 \u0634\u064a.<\/li>\n - \u0633\u062a\u0627\u0633\u0648 \u062f \u062d\u062c\u0631\u0648 \u0634\u0645\u06cc\u0631\u0647 \u0645\u062d\u0627\u0633\u0628\u0647 \u06a9\u0648\u0644\u0648 \u0648\u0631\u0648\u0633\u062a\u0647\u060c \u067e\u0647 2.5 x 10 \u06a9\u06d0 \u062d\u062c\u0631\u06d0 \u067e\u0644\u06cc\u067c \u06a9\u0693\u06265<\/sup>\u00a0<\/span>\u062d\u062c\u0631\u06d0 \u067e\u0647 \u0647\u0631 T25 \u0641\u0644\u067c\u0631 \u067c\u0627\u067e \u0641\u0644\u0627\u0633\u06a9 \u06a9\u06d0.<\/li>\n
- \u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u067e\u0647 \u0647\u0631\u0648 2-3 \u0648\u0631\u0681\u0648 \u06a9\u06d0 \u062f \u062a\u0627\u0632\u0647 \u0648\u062f\u06d0 \u0645\u0646\u0681\u0646\u064a \u0633\u0631\u0647 \u062a\u063a\u0630\u06cc\u0647 \u0634\u064a.<\/li>\n<\/ol>\n
\u06cc\u062e \u06a9\u0648\u0644:
<\/u><\/strong><\/h4>\n\u062d\u062c\u0631\u06d0 \u0628\u0627\u06cc\u062f \u0644\u0647 5 x 10 \u0685\u062e\u0647 \u06a9\u0645 \u0646\u0647 \u0648\u064a \u0645\u0646\u062c\u0645\u062f \u0634\u064a5<\/sup>\u00a0<\/span>\u062d\u062c\u0631\u06d0\/ml\/cryovial \u062f \u0648\u062f\u06d0 \u0645\u06cc\u0689\u06cc\u0627 \u06a9\u06d0 \u0686\u06d0 10% DMSO \u0627\u0648 30% FBS \u0644\u0631\u064a \u0627\u0648 \u0648\u0631\u0648\u0633\u062a\u0647 \u062f \u0634\u067e\u06d0 \u067e\u0647 -80\u02daC \u06a9\u06d0 \u062f isopropanol \u067e\u0647 \u06cc\u062e\u0686\u0627\u0644 \u06a9\u06d0 \u0681\u0627\u06cc \u067e\u0647 \u0681\u0627\u06cc \u0634\u0648\u064a. \u0628\u0644\u0647 \u0648\u0631\u0681 \u0645\u0627\u06cc\u0639 \u0646\u0627\u06cc\u067c\u0631\u0648\u062c\u0646 \u062a\u0647 \u0627\u0646\u062a\u0642\u0627\u0644 \u06a9\u0693\u0626.<\/p>\n\u062f \u0641\u0631\u0639\u064a \u06a9\u0644\u062a\u0648\u0631 \u06a9\u0648\u0644\u0648 \u0627\u0648 \u0645\u0646\u062c\u0645\u062f \u0641\u06cc\u0628\u0631\u0648\u0628\u0644\u0627\u0633\u067c\u0648\u0646\u0648 \u067e\u0631\u0648\u062a\u0648\u06a9\u0648\u0644<\/a><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_tablet=\u201d|35px||35px||\u0631\u06cc\u069a\u062a\u06cc\u0627\u201d custom_padding_phone=\u201d\u201dborder_width_top=\u201d10px\u201dborder_color_top=\u201d#8fd2ed\u201d \u0628\u0646\u062f =\u201d\u0628\u0646\u062f\u201d Global_colors_info=\u201d{}\u201d][et_pb_column_info="{}"][et_pb_column_info="_inner_type_4_Seave" _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d Global_colors_info="{}" custom_padding__hover="|||"][et_pb_text _builder_version="4.16\u2033 global_colors_info="{}"]<\/p>\n
\u062f \u0646\u0648\u0631\u0648 \u0645\u0639\u0644\u0648\u0645\u0627\u062a\u0648 \u0644\u067e\u0627\u0631\u0647 \u0645\u0647\u0631\u0628\u0627\u0646\u064a \u0648\u06a9\u0693\u0626 \u0627\u0693\u06cc\u06a9\u0647 \u0648\u0646\u06cc\u0633\u0626:<\/strong><\/h4>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\u201d on|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d|35px|0|0px|\u063a\u0644\u0637 ="1_2\u2033 \u062e\u0648\u0646\u062f\u064a \u0634\u0648\u06cc_\u062e\u0627\u0635\u06cc\u062a_\u06a9\u0627\u0644\u0645_\u0689\u0648\u0644 =\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|20px||\u201d custom_padding_tablet="||20px" custom_padding_phone="" custom_padding_last_edited="on|desktop" global_colors_info="{}" custom_padding__hover="|||"][et_pb_text _builder_version="4.19.5\u2033 \u067e\u0647 \u0646\u0693\u06cc\u0648\u0627\u0644 \u06a9\u06d0"}_colors"}<\/p>\n
\u0644\u06cc\u0633\u0644\u064a \u0628\u06cc \u06ab\u0648\u0631\u0689\u0646\u060c MD\u060c PhD<\/h6>\n
\u062f \u0628\u0631\u0627\u0648\u0646 \u067e\u0648\u0647\u0646\u062a\u0648\u0646 \u062f \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u062f \u0685\u06cc\u0693\u0646\u06d0 \u067e\u0631\u0648\u0641\u06cc\u0633\u0648\u0631 \u0648\u0627\u0631\u0646 \u0627\u0644\u067e\u0631\u067c \u0637\u0628\u064a \u069a\u0648\u0648\u0646\u0681\u064a \u0627\u0648 \u062f \u067e\u06cc\u0689\u06cc\u0627\u067c\u0631\u06cc\u06a9\u0633 \u0689\u06cc\u067e\u0627\u0631\u067c\u0645\u0646\u067c \u060c \u062f \u0647\u0627\u0633\u0628\u0631\u0648 \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u0631\u0648\u063a\u062a\u0648\u0646 \u060c \u067e\u0631\u0648\u0648\u06cc\u0689\u0646\u0633 \u060c \u062f \u0627\u0646\u0633\u062a\u06cc\u0632\u06cc\u0627 RI \u0685\u0627\u0646\u06ab\u0647 \u060c \u062f \u0645\u0627\u0634\u0648\u0645\u0627\u0646\u0648 \u0631\u0648\u063a\u062a\u0648\u0646 \u0628\u0648\u0633\u067c\u0646 \u0627\u0648 \u0647\u0627\u0631\u0648\u0631\u0689 \u0645\u06cc\u0689\u06cc\u06a9\u0644 \u069a\u0648\u0648\u0646\u0681\u06cc \u060c \u0628\u0648\u0633\u067c\u0646 \u060c \u062f MA \u0637\u0628\u064a \u0631\u06cc\u06cc\u0633 \u060c \u062f \u067e\u0631\u0648\u062c\u06cc\u0631\u06cc\u0627 \u0631\u06cc\u0633\u0631\u0686 \u0641\u0627\u0648\u0646\u0689\u06cc\u0634\u0646<\/p>\n
\u062a\u0644\u06cc\u0641\u0648\u0646: 978-535-2594
\u0641\u06a9\u0633: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n] custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|phone\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_last_edited=\u201doff|desktop\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033]<\/p>\n
\u0648\u06cc\u0646\u0689\u064a \u0646\u0648\u0631\u06cc\u0633<\/span><\/h6>\n\u062f PRF \u0633\u06cc\u0644 \u0627\u0648 \u0646\u0633\u062c \u0628\u0627\u0646\u06a9
\u062a\u0644\u06cc\u0641\u0648\u0646: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
<\/span><\/div>\n\u062f custom_margin="-2px||||||" custom_padding=\u201d0|0px|0|0px|\u063a\u0644\u0637 column_structure=\u201d1_3,1_3,1_3\u2033 disabled_on=\u201don|on|on\u201d admin_label=\u201dRow\u201d _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d min_height=\u201d277px\u201d custom_margin=\u201d|20pxal|20pxal | \u062f\u0631\u0648\u063a" custom_padding="0px|30px|0px||false|false" disabled="on"global_colors_info="{}"][et_pb_column_inner type="1_3\u2033 saved_specialty_column_type="3_4\u2033 _builder\u2033_version="_preet ="4" _builder_version="4. global_colors_info="{}"][et_pb_image src="https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/08\/Startbanner-1.png" title_text="Startbanner" url="https:\/\/ www.gravoc.com\/\u201d align=\u201dright\u201d _builder_version=\u201d4.21.0\u2033 _module_preset="default" custom_margin="|-50px||20px|false|false" global_colors_info="{}"][\/et_pb_image][\/et_pb_column_inner][et_pb_column_inner type="1_3\u2033column_inner type="1_3\u2033column_type="1_3\u2033 type_4" \u062e\u0648\u0646\u062f\u064a \u0634\u0648\u06cc _builder_version="4.21.0\u2033 _module_preset="default" global_colors_info="{}"][\/et_pb_column_inner][et_pb_column_inner type="1_3\u2033 saved_specialty_column_type="3\u2033_4\u2033 ion = 1_4\u2033 _builder _4\u2033. _module_preset="default" global_colors_info="{}"][et_pb_image src="https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/07\/RFRFinish.png" title_text="RFRFinish" _builder_vers. .0\u2033 _module_preset="default" custom_margin="|20px||-50px|\u063a\u0644\u0637 make_equal = "\u067e\u0647" \u0645\u0648\u0689\u0644_\u06a9\u0644\u0627\u0633 =" et_pb_row_fullwidth" _builder_version="4.16" \u0639\u0631\u0636 = "89%" width_tablet="80%" width_phone="" width_last_edited="on"|Tip_9th="max_9th" max_width_tablet=\u201d80%\u201d max_width_phone=\u201d\u201dmax_width_last_edited="on|desktop\u201d z_index_tablet=\u201d500\u2033 make_fullwidth="on" width_unit="off" custom_width_percent=\u201dT301 Global_colors_info=\u201d{}\u201d][et_pb_column type=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info="{}" custom_padding__hover="|||"][et_pb_cta button_url="https:\/\/www.progeriaresearch.org\/newsletter-signup\/" button_text="\u0627\u0648\u0633 \u0646\u0648\u0645 \u0644\u06cc\u06a9\u0646\u0647 \u0648\u06a9\u0693\u0626" admin_label="\u062f \u062e\u0628\u0631 \u067e\u0627\u06bc\u064a \u0644\u067e\u0627\u0631\u0647 \u0644\u0627\u0633\u0644\u06cc\u06a9 \u0648\u06a9\u0693\u0626" \u0645\u0627\u0689\u0644_\u06a9\u0644\u0627\u0633 ="sign-btn" _builder_version="4.16\u2033 header_font_size=\u201d25px\u201d background_color=\u201d#29327a\u201d animation_style=\u201dslide\u201d animation_direction=\u201dleft\u201d animation_intensity_slide=\u201d25%\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d30p header_font_size_last_edited=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 Border_radii=\u201don|25px|25px|25px|25px\u201d Global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201don\u201dcolor_tverg=\u201dtp__tverg1\u201don\u201d. button_border_color__hover_enabled=\u201d\u0686\u0627\u0644\u0648\u201d]<\/p>\n
\u06ab\u0689\u0648\u0646 \u06a9\u0648\u0644<\/h2>\n\u0632\u0645\u0648\u0696 \u0644\u067e\u0627\u0631\u0647<\/h2>\n\u062e\u0628\u0631\u067e\u0627\u06bc\u0647!<\/h2>\n
[\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info="{}" custom_padding__hover="|||"][et_pb_cta button_url="https:\/\/progeriaresearch.donorsupport.co\/-\/XZHJVWZR" button_text="\u0627\u0648\u0633 \u0645\u0631\u0633\u062a\u0647 \u0648\u06a9\u0693\u0626" admin_label="\u067e\u0647 \u06ab\u0689\u0647\u060c \u0645\u0648\u0696 \u0628\u0647 \u062f\u0631\u0645\u0644\u0646\u0647 \u0648\u0645\u0648\u0645\u0626! \u00bb module_class = "sign-btn" _builder_version="4.16" header_font_size="25px" background_color="#29327a" animation_style="slide" animation_direction="left" animation_intensity_slide="T_font_slide="T_font_size="251 \u067c\u0627\u0628\u0644\u06cc\u067c header_font_size_phone="30px" header_font_size_last_edited="on|desktop" body_font_size_tablet="" body_font_size_phone="" body_font_size_last_edited="on|desktop" z_index_tablet="500\u2033 border_radii=\u201don|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201don\u201d button_bg_color__hover=\u201d#8fd2ed\u201d button_border_color__hover_enabled=\u201d]<\/p>\n
\u067e\u0647 \u06ab\u0689\u0647\u060c \u0645\u0648\u0696<\/h2>\n\u0628\u0647<\/em><\/h2>\n\u0639\u0644\u0627\u062c \u067e\u06cc\u062f\u0627 \u06a9\u0693\u0647!<\/h2>\n
[\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info="{}" custom_padding__hover="|||"][et_pb_image src="https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2024\/08\/2024-strip-footer-strip-copy.png "\u0633\u0631\u0644\u06cc\u06a9_\u0645\u062a\u0646 ="2024 \u067e\u067c\u06d0 \u0641\u0648\u067c\u0631 \u067e\u067c\u06d0 \u06a9\u0627\u067e\u064a" _builder_version="4.27.0\u2033 _module_preset="default" custom_margin="35px||||false|false" global_colors_info="{}"][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_ction]<\/p>","protected":false},"excerpt":{"rendered":"
[et_pb_section fb_built=”1″ fullwidth=”on” disabled_on=”off|off|off” _builder_version=”4.16″ border_width_bottom=”55px” border_color_bottom=”#29327a” locked=”off” global_colors_info=”{}”][et_pb_fullwidth_header _builder_version=”4.16″ title_font=”||||||||” title_font_size=”55″ background_color=”#29327a” background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg” background_position=”center_left” custom_padding=”9vw||9vw||true” custom_padding_tablet=”” custom_padding_phone=”|56px||” custom_padding_last_edited=”on|desktop” title_font_size_tablet=”45px” title_font_size_phone=”40px” title_font_size_last_edited=”on|phone” z_index_tablet=”500″ custom_css_main_element=”background-position: center 18% !important;” global_colors_info=”{}”] Fibroblast Cell Culture Protocols [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” […]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Fibroblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n
PROTOCOL FOR SUBCULTURING AND FREEZING FIBROBLASTS\u00a0<\/strong><\/p>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=\"1\/2\"][vc_column_text]Growth Media<\/u><\/strong>\r\n\r\nDMEM - ThermoFisher #11960-044 (high glucose without L-glutamine)\r\n\r\n15% FBS Fetal Bovine Serum \u2013 ThermoFisher #10437-028\r\n\r\n1% (1X) Penicillin-Streptomycin - ThermoFisher #15140-122\r\n\r\n1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061[\/vc_column_text][\/vc_column][vc_column width=\"1\/2\"][vc_column_text]Trypsin<\/u><\/strong>\r\n\r\nTrypsin EDTA C 0.25% (ThermoFisher #25200-056)\r\n\r\nHank\u2019s Balanced Salt Solution\r\n<\/u><\/strong>HBSS- (ThermoFisher #14170 (1X) (-) calcium chloride, (-) magnesium chloride, (-) magnesium sulfate\r\n\r\nDMSO for Freezing\r\n<\/u><\/strong>Cell Culture Grade DMSO - Sigma #D2438[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Thawing PRF cell lines<\/u><\/strong>\r\n\r\n \t- Thaw cells rapidly in a 37\u02daC water bath.<\/li>\r\n \t
- Wipe the outside of the vial with 70% ethanol.<\/li>\r\n \t
- Transfer the thawed cells to a T25 flask containing 5 ml of growth media.<\/li>\r\n \t
- Place flask in 37\u02daC incubator overnight.<\/li>\r\n \t
- The following day, observe under the microscope to ensure cells have attached.<\/li>\r\n \t
- Remove media and replace with fresh growth media.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Subculturing PRF cell lines<\/u><\/strong>\r\n
\r\n \t- Cells should be split when confluent.<\/li>\r\n \t
- To split cells (volumes given are assuming cells are in a T25):\r\na)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.\r\nb)\u00a0 Add 1 ml trypsin and incubate for 2-3 minutes. Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float.\u00a0 If cells are not detached after 3 minutes, you may incubate another 1-2 minutes.\r\nc)\u00a0 Tighten cap and gently tip flask from side to side to dislodge all cells. Flask may be tapped lightly on side to loosen cells if needed.\r\nd)\u00a0 Add 4 ml of new media to flask as soon as cells are floating to inactivate the trypsin. Rinse down sides of flask several times with the new media to wash all cells off the plastic and into the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).\r\ne)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.\r\nf)\u00a0 While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpm.<\/li>\r\n \t
- After calculating your cell count, plate cells at 2.5 x 105<\/sup> cells per T25 filter top flask.<\/li>\r\n \t
- Cells should be fed every 2-3 days with fresh growth medium.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Freezing:\r\n<\/u><\/strong>Cells should be frozen at no less than 5 x 105<\/sup> cells\/ml\/cryovial in growth media containing 10% DMSO and 30% FBS and subsequently placed in an isopropanol freezing chamber at -80\u02daC overnight.\u00a0 Transfer to the liquid nitrogen the next day.\r\n\r\n\u00a0Protocol for Subculturing and Freezing Fibroblasts<\/a>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA,\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n
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\u0644\u06cc\u0633\u0644\u064a \u0628\u06cc \u06ab\u0648\u0631\u0689\u0646\u060c MD\u060c PhD<\/h6>\n
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\u062a\u0644\u06cc\u0641\u0648\u0646: 978-535-2594
\u0641\u06a9\u0633: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n] custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|phone\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_last_edited=\u201doff|desktop\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033]<\/p>\n
\u0648\u06cc\u0646\u0689\u064a \u0646\u0648\u0631\u06cc\u0633<\/span><\/h6>\n\u062f PRF \u0633\u06cc\u0644 \u0627\u0648 \u0646\u0633\u062c \u0628\u0627\u0646\u06a9
\u062a\u0644\u06cc\u0641\u0648\u0646: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
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[\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info="{}" custom_padding__hover="|||"][et_pb_image src="https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2024\/08\/2024-strip-footer-strip-copy.png "\u0633\u0631\u0644\u06cc\u06a9_\u0645\u062a\u0646 ="2024 \u067e\u067c\u06d0 \u0641\u0648\u067c\u0631 \u067e\u067c\u06d0 \u06a9\u0627\u067e\u064a" _builder_version="4.27.0\u2033 _module_preset="default" custom_margin="35px||||false|false" global_colors_info="{}"][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_ction]<\/p>","protected":false},"excerpt":{"rendered":"
[et_pb_section fb_built=”1″ fullwidth=”on” disabled_on=”off|off|off” _builder_version=”4.16″ border_width_bottom=”55px” border_color_bottom=”#29327a” locked=”off” global_colors_info=”{}”][et_pb_fullwidth_header _builder_version=”4.16″ title_font=”||||||||” title_font_size=”55″ background_color=”#29327a” background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg” background_position=”center_left” custom_padding=”9vw||9vw||true” custom_padding_tablet=”” custom_padding_phone=”|56px||” custom_padding_last_edited=”on|desktop” title_font_size_tablet=”45px” title_font_size_phone=”40px” title_font_size_last_edited=”on|phone” z_index_tablet=”500″ custom_css_main_element=”background-position: center 18% !important;” global_colors_info=”{}”] Fibroblast Cell Culture Protocols [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” […]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Fibroblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n
PROTOCOL FOR SUBCULTURING AND FREEZING FIBROBLASTS\u00a0<\/strong><\/p>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=\"1\/2\"][vc_column_text]Growth Media<\/u><\/strong>\r\n\r\nDMEM - ThermoFisher #11960-044 (high glucose without L-glutamine)\r\n\r\n15% FBS Fetal Bovine Serum \u2013 ThermoFisher #10437-028\r\n\r\n1% (1X) Penicillin-Streptomycin - ThermoFisher #15140-122\r\n\r\n1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061[\/vc_column_text][\/vc_column][vc_column width=\"1\/2\"][vc_column_text]Trypsin<\/u><\/strong>\r\n\r\nTrypsin EDTA C 0.25% (ThermoFisher #25200-056)\r\n\r\nHank\u2019s Balanced Salt Solution\r\n<\/u><\/strong>HBSS- (ThermoFisher #14170 (1X) (-) calcium chloride, (-) magnesium chloride, (-) magnesium sulfate\r\n\r\nDMSO for Freezing\r\n<\/u><\/strong>Cell Culture Grade DMSO - Sigma #D2438[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Thawing PRF cell lines<\/u><\/strong>\r\n\r\n \t- Thaw cells rapidly in a 37\u02daC water bath.<\/li>\r\n \t
- Wipe the outside of the vial with 70% ethanol.<\/li>\r\n \t
- Transfer the thawed cells to a T25 flask containing 5 ml of growth media.<\/li>\r\n \t
- Place flask in 37\u02daC incubator overnight.<\/li>\r\n \t
- The following day, observe under the microscope to ensure cells have attached.<\/li>\r\n \t
- Remove media and replace with fresh growth media.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Subculturing PRF cell lines<\/u><\/strong>\r\n
\r\n \t- Cells should be split when confluent.<\/li>\r\n \t
- To split cells (volumes given are assuming cells are in a T25):\r\na)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.\r\nb)\u00a0 Add 1 ml trypsin and incubate for 2-3 minutes. Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float.\u00a0 If cells are not detached after 3 minutes, you may incubate another 1-2 minutes.\r\nc)\u00a0 Tighten cap and gently tip flask from side to side to dislodge all cells. Flask may be tapped lightly on side to loosen cells if needed.\r\nd)\u00a0 Add 4 ml of new media to flask as soon as cells are floating to inactivate the trypsin. Rinse down sides of flask several times with the new media to wash all cells off the plastic and into the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).\r\ne)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.\r\nf)\u00a0 While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpm.<\/li>\r\n \t
- After calculating your cell count, plate cells at 2.5 x 105<\/sup> cells per T25 filter top flask.<\/li>\r\n \t
- Cells should be fed every 2-3 days with fresh growth medium.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Freezing:\r\n<\/u><\/strong>Cells should be frozen at no less than 5 x 105<\/sup> cells\/ml\/cryovial in growth media containing 10% DMSO and 30% FBS and subsequently placed in an isopropanol freezing chamber at -80\u02daC overnight.\u00a0 Transfer to the liquid nitrogen the next day.\r\n\r\n\u00a0Protocol for Subculturing and Freezing Fibroblasts<\/a>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA,\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n
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background_color=”#29327a” background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg” background_position=”center_left” custom_padding=”9vw||9vw||true” custom_padding_tablet=”” custom_padding_phone=”|56px||” custom_padding_last_edited=”on|desktop” title_font_size_tablet=”45px” title_font_size_phone=”40px” title_font_size_last_edited=”on|phone” z_index_tablet=”500″ custom_css_main_element=”background-position: center 18% !important;” global_colors_info=”{}”] Fibroblast Cell Culture Protocols [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” […]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Fibroblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n PROTOCOL FOR SUBCULTURING AND FREEZING FIBROBLASTS\u00a0<\/strong><\/p>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=\"1\/2\"][vc_column_text]Growth Media<\/u><\/strong>\r\n\r\nDMEM - ThermoFisher #11960-044 (high glucose without L-glutamine)\r\n\r\n15% FBS Fetal Bovine Serum \u2013 ThermoFisher #10437-028\r\n\r\n1% (1X) Penicillin-Streptomycin - ThermoFisher #15140-122\r\n\r\n1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061[\/vc_column_text][\/vc_column][vc_column width=\"1\/2\"][vc_column_text]Trypsin<\/u><\/strong>\r\n\r\nTrypsin EDTA C 0.25% (ThermoFisher #25200-056)\r\n\r\nHank\u2019s Balanced Salt Solution\r\n<\/u><\/strong>HBSS- (ThermoFisher #14170 (1X) (-) calcium chloride, (-) magnesium chloride, (-) magnesium sulfate\r\n\r\nDMSO for Freezing\r\n<\/u><\/strong>Cell Culture Grade DMSO - Sigma #D2438[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Thawing PRF cell lines<\/u><\/strong>\r\n\u062f \u0646\u0648\u0631\u0648 \u0645\u0639\u0644\u0648\u0645\u0627\u062a\u0648 \u0644\u067e\u0627\u0631\u0647 \u0645\u0647\u0631\u0628\u0627\u0646\u064a \u0648\u06a9\u0693\u0626 \u0627\u0693\u06cc\u06a9\u0647 \u0648\u0646\u06cc\u0633\u0626:<\/strong><\/h4>\n
\u0644\u06cc\u0633\u0644\u064a \u0628\u06cc \u06ab\u0648\u0631\u0689\u0646\u060c MD\u060c PhD<\/h6>\n
\u0641\u06a9\u0633: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n\u0648\u06cc\u0646\u0689\u064a \u0646\u0648\u0631\u06cc\u0633<\/span><\/h6>\n
\u062a\u0644\u06cc\u0641\u0648\u0646: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
<\/span><\/div>\n\u06ab\u0689\u0648\u0646 \u06a9\u0648\u0644<\/h2>\n
\u0632\u0645\u0648\u0696 \u0644\u067e\u0627\u0631\u0647<\/h2>\n
\u062e\u0628\u0631\u067e\u0627\u06bc\u0647!<\/h2>\n
\u067e\u0647 \u06ab\u0689\u0647\u060c \u0645\u0648\u0696<\/h2>\n
\u0628\u0647<\/em><\/h2>\n
\u0639\u0644\u0627\u062c \u067e\u06cc\u062f\u0627 \u06a9\u0693\u0647!<\/h2>\n
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\u00a0Protocol for Subculturing and Freezing Fibroblasts<\/a>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA,\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n