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\u09b9\u09af\u09bc\u09c7 \u0997\u09c7\u09b2\u09c7, 10 \u03bcg\/ml \u09aa\u09b2\u09bf\u09ac\u09cd\u09b0\u09bf\u09a8\u09c7\u09b0 \u099a\u09c2\u09a1\u09bc\u09be\u09a8\u09cd\u09a4 \u0998\u09a8\u09a4\u09cd\u09ac\u09c7\u09b0 \u09b8\u09be\u09a5\u09c7 8 \u09ae\u09bf\u09b2\u09bf \u09ad\u09be\u0987\u09b0\u09be\u09b8\u09af\u09c1\u0995\u09cd\u09a4 \u09ae\u09be\u09a7\u09cd\u09af\u09ae \u09af\u09cb\u0997 \u0995\u09b0\u09be \u09b9\u09af\u09bc\u09c7\u099b\u09bf\u09b2\u0964 \u09b8\u0982\u0995\u09cd\u09b0\u09ae\u09a3\u09c7\u09b0 \u09a4\u09bf\u09a8\u099f\u09bf \u09b0\u09be\u0989\u09a8\u09cd\u09a1 \u0995\u09cd\u09b0\u09ae\u09be\u09a8\u09cd\u09ac\u09af\u09bc\u09c7 \u09b8\u099e\u09cd\u099a\u09be\u09b2\u09bf\u09a4 \u09b9\u09af\u09bc\u09c7\u099b\u09bf\u09b2, ~ 12 \u0998\u09a8\u09cd\u099f\u09be\u09b0 \u09ac\u09cd\u09af\u09ac\u09a7\u09be\u09a8\u09c7\u0964 24 \u0998\u09a8\u09cd\u099f\u09be \u0987\u09a8\u0995\u09bf\u0989\u09ac\u09c7\u09b6\u09a8\u09c7\u09b0 \u09aa\u09b0\u09c7 \u09ae\u09be\u09a7\u09cd\u09af\u09ae\u099f\u09bf \u09aa\u09cd\u09b0\u09a4\u09bf\u09b8\u09cd\u09a5\u09be\u09aa\u09bf\u09a4 \u09b9\u09af\u09bc\u09c7\u099b\u09bf\u09b2\u0964<\/p>\n<p><strong>\u0997. \u09b8\u09cd\u09a5\u09bf\u09a4\u09bf\u09b6\u09c0\u09b2 \u09b8\u09c7\u09b2 \u09b2\u09be\u0987\u09a8 \u09a8\u09bf\u09b0\u09cd\u09ac\u09be\u099a\u09a8:<\/strong> \u09b8\u0982\u0995\u09cd\u09b0\u09ae\u09a3\u09c7\u09b0 72 \u0998\u09a8\u09cd\u099f\u09be \u09aa\u09b0\u09c7, \u0995\u09cb\u09b7\u0997\u09c1\u09b2\u09bf\u0995\u09c7 \u099f\u09cd\u09b0\u09bf\u09aa\u09b8\u09bf\u09a8\u09be\u0987\u099c \u0995\u09b0\u09be \u09b9\u09af\u09bc, 1:3 \u098f \u09ac\u09bf\u09ad\u0995\u09cd\u09a4 \u0995\u09b0\u09be \u09b9\u09af\u09bc \u098f\u09ac\u0982 3 \u03bcg\/ml \u09aa\u09bf\u0989\u09b0\u09cb\u09ae\u09be\u0987\u09b8\u09bf\u09a8\u09c7\u09b0 \u0989\u09aa\u09b8\u09cd\u09a5\u09bf\u09a4\u09bf\u09a4\u09c7 \u09a6\u09c1\u099f\u09bf 100 \u09ae\u09bf\u09ae\u09bf \u09aa\u09cd\u09b2\u09c7\u099f\u09c7 \u09b8\u09cd\u09a5\u09be\u09a8\u09be\u09a8\u09cd\u09a4\u09b0\u09bf\u09a4 \u0995\u09b0\u09be \u09b9\u09af\u09bc, \u098f\u099b\u09be\u09a1\u09bc\u09be\u0993 \u0985\u09cd\u09af\u09be\u09a8\u09cd\u099f\u09bf\u09ac\u09be\u09af\u09bc\u09cb\u099f\u09bf\u0995 \u099b\u09be\u09a1\u09bc\u09be\u0987 \u098f\u0995\u099f\u09bf \u09a8\u09bf\u09af\u09bc\u09a8\u09cd\u09a4\u09cd\u09b0\u09a3 \u09aa\u09cd\u09b2\u09c7\u099f\u0964 \u09b8\u09cd\u09a5\u09bf\u09a4\u09bf\u09b6\u09c0\u09b2 \u0995\u09cb\u09b7 \u09b2\u09be\u0987\u09a8 \u09a4\u09c8\u09b0\u09bf \u0995\u09b0\u09a4\u09c7 \u09aa\u09cd\u09b0\u09be\u09af\u09bc 2 \u09a5\u09c7\u0995\u09c7 4 \u09b8\u09aa\u09cd\u09a4\u09be\u09b9\u09c7\u09b0 \u099c\u09a8\u09cd\u09af \u09aa\u09cd\u09b0\u09a4\u09bf 2 \u09ac\u09be 3 \u09a6\u09bf\u09a8\u09c7 \u09b8\u0982\u09b8\u09cd\u0995\u09c3\u09a4\u09bf\u09b0 \u09ae\u09be\u09a7\u09cd\u09af\u09ae \u09aa\u09b0\u09bf\u09ac\u09b0\u09cd\u09a4\u09a8 \u0995\u09b0\u09be \u09b9\u09af\u09bc\u09c7\u099b\u09bf\u09b2\u0964<\/p>\n<p>&nbsp;<\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=&#8221;1_2,1_2&#8243; custom_padding_last_edited=&#8221;on|phone&#8221; _builder_version=&#8221;4.16&#8243; custom_margin=&#8221;|||||&#8221; custom_padding=&#8221;0px|35px|0|0px|false|false&#8221; custom_padding_tablet=&#8221;|35px|20px|35px||true&#8221; custom_padding_phone=&#8221;|35px|11px|35px|false|true&#8221; animation_direction=&#8221;top&#8221; global_colors_info=&#8221;{}&#8221;][et_pb_column_inner type=&#8221;1_2&#8243; saved_specialty_column_type=&#8221;3_4&#8243; _builder_version=&#8221;4.16&#8243; custom_padding=&#8221;|20px||&#8221; custom_padding_tablet=&#8221;||20px&#8221; custom_padding_phone=&#8221;&#8221; custom_padding_last_edited=&#8221;on|desktop&#8221; global_colors_info=&#8221;{}&#8221; custom_padding__hover=&#8221;|||&#8221;][et_pb_text _builder_version=&#8221;4.16&#8243; background_size=&#8221;initial&#8221; background_position=&#8221;top_left&#8221; background_repeat=&#8221;repeat&#8221; custom_padding=&#8221;||24px|||&#8221; global_colors_info=&#8221;{}&#8221;]<\/p>\n<h4>\u0997\u09cd\u09b0\u09cb\u09a5 \u09ae\u09bf\u09a1\u09bf\u09af\u09bc\u09be<\/h4>\n<p><span>DMEM- \u0987\u09a8\u09ad\u09bf\u099f\u09cd\u09b0\u09cb\u099c\u09c7\u09a8 #11960-044 (\u098f\u09b2-\u0997\u09cd\u09b2\u09c1\u099f\u09be\u09ae\u09bf\u09a8 \u099b\u09be\u09a1\u09bc\u09be \u0989\u099a\u09cd\u099a \u0997\u09cd\u09b2\u09c1\u0995\u09cb\u099c)<\/span><\/p>\n<p><span>15% FBS \u09ab\u09c7\u099f\u09be\u09b2 \u09ac\u09cb\u09ad\u09be\u0987\u09a8 \u09b8\u09bf\u09b0\u09be\u09ae<\/span><\/p>\n<p><span>1% (1x) \u09aa\u09c7\u09a8\u09bf\u09b8\u09bf\u09b2\u09bf\u09a8-\u09b8\u09cd\u099f\u09cd\u09b0\u09c7\u09aa\u09cd\u099f\u09cb\u09ae\u09be\u0987\u09b8\u09bf\u09a8 (\u0987\u09a8\u09ad\u09bf\u099f\u09cd\u09b0\u09cb\u099c\u09c7\u09a8#15140-122)<\/span><\/p>\n<p><span>1% (1X) \u098f\u09b2-\u0997\u09cd\u09b2\u09c1\u099f\u09be\u09ae\u09bf\u09a8 (\u0987\u09a8\u09ad\u09bf\u099f\u09cd\u09b0\u09cb\u099c\u09c7\u09a8#25030-081)<\/span><\/p>\n<p><span>0.75 \u03bcg\/ml puromycin (InvivoGen #ant-pr-1)<\/span><\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||20px\u201d custom_padding_tablet=\u201d20px|||0px\u201d 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=\u201d4.16\u2033 background_size=\u201dinitial\u201d background_position=\u201dtop_left\u201d background_repeat=\u201drepeat\u201d custom_padding=\u201d||24px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n<h4><strong>\u099f\u09cd\u09b0\u09bf\u09aa\u09b8\u09bf\u09a8<\/strong><\/h4>\n<p><span>Trypsin EDTA C .25% (Invitrogen#25200-056)<\/span><\/p>\n<h4><strong>\u09b9\u09cd\u09af\u09be\u0999\u09cd\u0995\u09c7\u09b0 \u09b8\u09c1\u09b7\u09ae \u09b2\u09ac\u09a3 \u09b8\u09ae\u09be\u09a7\u09be\u09a8<\/strong><\/h4>\n<p><span>HBSS- (Invitrogen#14170 (1X) (-) \u0995\u09cd\u09af\u09be\u09b2\u09b8\u09bf\u09af\u09bc\u09be\u09ae \u0995\u09cd\u09b2\u09cb\u09b0\u09be\u0987\u09a1, (-)\u09ae\u09cd\u09af\u09be\u0997\u09a8\u09c7\u09b8\u09bf\u09af\u09bc\u09be\u09ae \u0995\u09cd\u09b2\u09cb\u09b0\u09be\u0987\u09a1, (-) \u09ae\u09cd\u09af\u09be\u0997\u09a8\u09c7\u09b8\u09bf\u09af\u09bc\u09be\u09ae \u09b8\u09be\u09b2\u09ab\u09c7\u099f)<\/span><\/p>\n<h4><strong>\u099c\u09ae\u09be\u099f \u09ac\u09be\u0981\u09a7\u09be\u09b0 \u099c\u09a8\u09cd\u09af DMSO<\/strong><\/h4>\n<p><span>\u09b8\u09c7\u09b2 \u0995\u09be\u09b2\u099a\u09be\u09b0 \u0997\u09cd\u09b0\u09c7\u09a1 DMSO<\/span><\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=&#8221;on|phone&#8221; _builder_version=&#8221;4.16&#8243; custom_margin=&#8221;15px||35px|||&#8221; custom_padding=&#8221;0px|35px|0|0px|false|false&#8221; custom_padding_tablet=&#8221;|35px|20px|35px||true&#8221; custom_padding_phone=&#8221;|35px|11px|35px|false|true&#8221; animation_direction=&#8221;top&#8221; global_colors_info=&#8221;{}&#8221;][et_pb_column_inner saved_specialty_column_type=&#8221;3_4&#8243; _builder_version=&#8221;4.16&#8243; custom_padding=&#8221;|20px||&#8221; custom_padding_tablet=&#8221;||20px&#8221; custom_padding_phone=&#8221;&#8221; custom_padding_last_edited=&#8221;on|desktop&#8221; global_colors_info=&#8221;{}&#8221; custom_padding__hover=&#8221;|||&#8221;][et_pb_text _builder_version=&#8221;4.16&#8243; custom_margin=&#8221;||35px&#8221; global_colors_info=&#8221;{}&#8221;]<\/p>\n<p><b>\u0995\u09cb\u09b7\u0997\u09c1\u09b2\u09bf \u09aa\u09cd\u09b0\u09be\u09af\u09bc 5 x 10 \u0998\u09a8\u09a4\u09cd\u09ac\u09c7 \u09b6\u09c1\u0995\u09a8\u09cb \u09ac\u09b0\u09ab\u09c7\u09b0 \u0989\u09aa\u09b0 \u09b9\u09bf\u09ae\u09be\u09af\u09bc\u09bf\u09a4 \u09b9\u09af\u09bc\u09c7 \u0986\u09b8\u09ac\u09c7<sup>5<\/sup>\u0995\u09cb\u09b7\/\u09b6\u09bf\u09b6\u09bf<\/b><\/p>\n<p>[\/et_pb_text][et_pb_text _builder_version=&#8221;4.16&#8243; background_size=&#8221;initial&#8221; background_position=&#8221;top_left&#8221; background_repeat=&#8221;repeat&#8221; custom_padding=&#8221;||0px|||&#8221; global_colors_info=&#8221;{}&#8221;]<\/p>\n<h4 data-fontsize=\"16\" data-lineheight=\"24\"><b>\u09ab\u09be\u0987\u09ac\u09cd\u09b0\u09cb\u09ac\u09cd\u09b2\u09be\u09b8\u09cd\u099f \u0997\u09b2\u09be\u09a8\u09cb\u09b0 \u099c\u09a8\u09cd\u09af \u09aa\u09cd\u09b0\u09cb\u099f\u09cb\u0995\u09b2<\/b><\/h4>\n<ol>\n<li>37 \u09a1\u09bf\u0997\u09cd\u09b0\u09bf \u09b8\u09c7\u09b2\u09b8\u09bf\u09af\u09bc\u09be\u09b8 \u099c\u09b2\u09c7\u09b0 \u09b8\u09cd\u09a8\u09be\u09a8\u09c7 \u0995\u09cb\u09b7\u0997\u09c1\u09b2\u09bf\u0995\u09c7 \u09a6\u09cd\u09b0\u09c1\u09a4 \u0997\u09b2\u09bf\u09af\u09bc\u09c7 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\u09b9\u09bf\u09ae\u09be\u09af\u09bc\u09bf\u09a4 \u0995\u09b0\u09be \u0989\u099a\u09bf\u09a4<sup>5<\/sup><span>\u00a0<\/span>\u0995\u09cb\u09b7 \/ml\/cryovial\u00a0<span>\u09ac\u09c3\u09a6\u09cd\u09a7\u09bf\u09b0 \u09ae\u09bf\u09a1\u09bf\u09af\u09bc\u09be\u09a4\u09c7<\/span><span>\u00a0<\/span>10% DMSO \u098f\u09ac\u0982 30% FBS \u09b0\u09af\u09bc\u09c7\u099b\u09c7\u09f7<span>\u00a0<\/span><strong>\u09aa\u09bf\u0989\u09b0\u09cb\u09ae\u09be\u0987\u09b8\u09bf\u09a8 \u099b\u09be\u09a1\u09bc\u09be<\/strong><span>\u00a0<\/span>\u098f\u09ac\u0982 \u09aa\u09b0\u09ac\u09b0\u09cd\u09a4\u09c0\u09a4\u09c7 \u09b0\u09be\u09a4\u09be\u09b0\u09be\u09a4\u09bf -80 \u09a1\u09bf\u0997\u09cd\u09b0\u09bf \u09b8\u09c7\u09b2\u09b8\u09bf\u09af\u09bc\u09be\u09b8 \u09a4\u09be\u09aa\u09ae\u09be\u09a4\u09cd\u09b0\u09be\u09af\u09bc \u098f\u0995\u099f\u09bf \u0986\u0987\u09b8\u09cb\u09aa\u09cd\u09b0\u09cb\u09aa\u09cd\u09af\u09be\u09a8\u09b2 \u09ab\u09cd\u09b0\u09bf\u099c\u09bf\u0982 \u099a\u09c7\u09ae\u09cd\u09ac\u09be\u09b0\u09c7 \u09b0\u09be\u0996\u09be \u09b9\u09af\u09bc\u0964 \u09aa\u09b0\u09c7\u09b0 \u09a6\u09bf\u09a8 \u09a4\u09b0\u09b2 \u09a8\u09be\u0987\u099f\u09cd\u09b0\u09cb\u099c\u09c7\u09a8\u09c7 \u09b8\u09cd\u09a5\u09be\u09a8\u09be\u09a8\u09cd\u09a4\u09b0 \u0995\u09b0\u09c1\u09a8\u0964<\/p>\n<p>[\/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||true\u201d custom_padding_phone=\u201d\u201d border_width_top=\u201d10px\u201d border_color_top=\u201d#8fd2ed\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.16\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n<h4><strong>\u0986\u09b0\u0993 \u09a4\u09a5\u09cd\u09af\u09c7\u09b0 \u099c\u09a8\u09cd\u09af \u0985\u09a8\u09c1\u0997\u09cd\u09b0\u09b9 \u0995\u09b0\u09c7 \u09af\u09cb\u0997\u09be\u09af\u09cb\u0997 \u0995\u09b0\u09c1\u09a8:<\/strong><\/h4>\n<p>[\/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=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d|35px|0|0px|false|false\u201d custom_padding_tablet=\u201d|35px||35px||true\u201d custom_padding_phone=\u201d\u201d animation_direction=\u201dtop\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|20px||\u201d custom_padding_tablet=\u201d||20px\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|desktop\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.19.5\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n<h6>\u09b2\u09c7\u09b8\u09b2\u09bf \u09ac\u09bf \u0997\u09b0\u09cd\u09a1\u09a8, \u098f\u09ae\u09a1\u09bf, \u09aa\u09bf\u098f\u0987\u099a\u09a1\u09bf<\/h6>\n<p>\u09ac\u09cd\u09b0\u09be\u0989\u09a8 \u0987\u0989\u09a8\u09bf\u09ad\u09be\u09b0\u09cd\u09b8\u09bf\u099f\u09bf\u09b0 \u09aa\u09c7\u09a1\u09bf\u09af\u09bc\u09be\u099f\u09cd\u09b0\u09bf\u0995\u09cd\u09b8 \u09b0\u09bf\u09b8\u09be\u09b0\u09cd\u099a \u0993\u09af\u09bc\u09be\u09b0\u09c7\u09a8 \u0985\u09cd\u09af\u09be\u09b2\u09aa\u09be\u09b0\u09cd\u099f \u09ae\u09c7\u09a1\u09bf\u0995\u09c7\u09b2 \u09b8\u09cd\u0995\u09c1\u09b2 \u098f\u09ac\u0982 \u09aa\u09c7\u09a1\u09bf\u09af\u09bc\u09be\u099f\u09cd\u09b0\u09bf\u0995\u09cd\u09b8 \u09ac\u09bf\u09ad\u09be\u0997\u09c7\u09b0 \u0985\u09a7\u09cd\u09af\u09be\u09aa\u0995, \u09b9\u09be\u09b8\u09ac\u09cd\u09b0\u09cb \u099a\u09bf\u09b2\u09a1\u09cd\u09b0\u09c7\u09a8 \u09b9\u09be\u09b8\u09aa\u09be\u09a4\u09be\u09b2, \u09aa\u09cd\u09b0\u09ad\u09bf\u09a1\u09c7\u09a8\u09cd\u09b8, \u0985\u09cd\u09af\u09be\u09a8\u09c7\u09b8\u09cd\u09a5\u09c7\u09b6\u09bf\u09af\u09bc\u09be\u09b0 RI \u09ac\u09bf\u09ad\u09be\u0997, \u09b6\u09bf\u09b6\u09c1 \u09b9\u09be\u09b8\u09aa\u09be\u09a4\u09be\u09b2 \u09ac\u09cb\u09b8\u09cd\u099f\u09a8 \u098f\u09ac\u0982 \u09b9\u09be\u09b0\u09cd\u09ad\u09be\u09b0\u09cd\u09a1 \u09ae\u09c7\u09a1\u09bf\u0995\u09c7\u09b2 \u09b8\u09cd\u0995\u09c1\u09b2, \u09ac\u09cb\u09b8\u09cd\u099f\u09a8, \u098f\u09ae\u098f \u09ae\u09c7\u09a1\u09bf\u0995\u09c7\u09b2 \u09a1\u09bf\u09b0\u09c7\u0995\u09cd\u099f\u09b0, \u09aa\u09cd\u09b0\u09cb\u099c\u09c7\u09b0\u09bf\u09af\u09bc\u09be \u09b0\u09bf\u09b8\u09be\u09b0\u09cd\u099a \u09ab\u09be\u0989\u09a8\u09cd\u09a1\u09c7\u09b6\u09a8<\/p>\n<p>\u09ab\u09cb\u09a8: 978-535-2594<br \/>\u09ab\u09cd\u09af\u09be\u0995\u09cd\u09b8: 508-543-0377<br \/><span> <\/span><a href=\"mailto:lgordon@progeriaresearch.org\" title=\"mailto:lgordon@progeriaresearch.org\">lgordon@progeriaresearch.org<\/a><\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||20px\u201d custom_padding=\u201d| 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<h6><span>\u0993\u09af\u09bc\u09c7\u09a8\u09cd\u09a1\u09bf \u09a8\u09b0\u09bf\u09b8<\/span><\/h6>\n<div><span>\u09aa\u09bf\u0986\u09b0\u098f\u09ab \u09b8\u09c7\u09b2 \u098f\u09ac\u0982 \u099f\u09bf\u09b8\u09cd\u09af\u09c1 \u09ac\u09cd\u09af\u09be\u0982\u0995<br \/>\u09ab\u09cb\u09a8: 401-274-1122 x 48063<br \/><a href=\"mailto:wnorris@brownhealth.org\" title=\"mailto:wnorris@brownhealth.org\" data-outlook-id=\"6ae643af-3829-46f7-8b6a-2ef767034802\">wnorris@brownhealth.org<\/a><a href=\"mailto:wnorris@lifespan.org\" target=\"_blank\" rel=\"noopener noreferrer\"><\/a><\/span><\/div>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 module_class=\u201dfooter\u201d _builder_version=\u2033TP_27=\u201d9.27\u201d \u09ac\u09be \u09aa\u099f\u09ad\u09c2\u09ae\u09bf\u201d4.21\u201dT21=20. custom_margin=&quot;-2px|||||&quot; custom_padding=\u201d0|0px|0|0px|false|false\u201d z_index_tablet=\u201d500\u2033 border_width_top=\u201d12px\u201d border_color_top=\u201d#00b2e2\u2033 global_module=\u201d133\u2033 locked=\u201doff\u201d global_colors_info=\u201d][_colors_info] 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|20px|se|20pxal |\u09ae\u09bf\u09a5\u09cd\u09af\u09be&quot; custom_padding=\u201d0px|30px|0px||false|false\u201d disabled=\u201don\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner type=\u201d1_3\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder\u2033_version=\u201d_deule=\u201d4.\u201d_\u09ac\u09bf\u09b2\u09cd\u09a1\u09be\u09b0\u2033 \u09b8\u09c7\u099f = 0 \u09aa\u09bf\u0995\u09cd\u09b8\u09c7\u09b2=\u201d4\u0964 global_colors_info=\u201d{}\u201d][et_pb_image src=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/08\/Startbanner-1.png\u201d title_text=\u201dStartbanner\u201d url=\u201dhttps:\/\/ www.gravoc.com\/\u201d align=\u201dright\u201d _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d custom_margin=\u201d|-50px||20px|false|false\u201d global_colors_info=\u201d{}\u201d][\/et_pb_image][\/et_pb_column_inner][et_pb_column_inner type=\u201d1_3\u2033type_3\u2033type_3\u2033type_s_3\u2033type_s_3\u2033 \u09b8\u09c7\u09ad \u0995\u09b0\u09be \u09b9\u09af\u09bc\u09c7\u099b\u09c7 _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d global_colors_info=\u201d{}\u201d][\/et_pb_column_inner][et_pb_column_inner type=\u201d1_3\u2033 saved_specialty_column_type=\u201d3\u2033_4\u2033 ion\u0964 _module_preset=\u201ddefault\u201d global_colors_info=\u201d{}\u201d][et_pb_image src=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/07\/RFRFinish.png\u201d title_text=\u201dRFRFinish\u201d _builder=4.2vers .0\u2033 _module_preset=&quot;default&quot; custom_margin=&quot;|20px||-50px|false|false&quot; global_colors_info=&quot;{}&quot;][\/et_pb_image][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row\u2033 \u0995\u09b2\u09be\u09ae, 1_1=4, 1_1= make_equal=&quot;on&quot; 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\u09b8\u09be\u0987\u09a8 \u0986\u09aa \u0995\u09b0\u09c1\u09a8\u201d \u09ae\u09a1\u09bf\u0989\u09b2_\u0995\u09cd\u09b2\u09be\u09b8 =\u201d\u09b8\u09be\u0987\u09a8-\u09ac\u09bf\u099f\u09bf\u098f\u09a8\u201d _\u09ac\u09bf\u09b2\u09cd\u09a1\u09be\u09b0_\u09b8\u0982\u09b8\u09cd\u0995\u09b0\u09a3=\u201d4.16\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=&quot;\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\u201d \u09ac\u09be TP_2f2\u201d \u09ac\u09be\u099f\u09a8 1 \u099f\u09bf\u09aa\u09bf button_border_color__hover_enabled=&quot;\u099a\u09be\u09b2\u09c1&quot;]<\/p>\n<h2>\u09b8\u09be\u0987\u09a8 \u0986\u09aa \u0995\u09b0\u09c1\u09a8<\/h2>\n<h2>\u0986\u09ae\u09be\u09a6\u09c7\u09b0 \u099c\u09a8\u09cd\u09af<\/h2>\n<h2>\u09a8\u09bf\u0989\u099c\u09b2\u09c7\u099f\u09be\u09b0 !<\/h2>\n<p>[\/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=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_cta button_url=\u201dhttps:\/\/progeriaresearch.donorsupport.co\/-\/XZHJVWZR\u201d button_text=\u201d\u098f\u0996\u09a8\u0987 \u09a6\u09be\u09a8 \u0995\u09b0\u09c1\u09a8\u201d admin_label=\u201d\u098f\u0995\u09b8\u09be\u09a5\u09c7, \u0986\u09ae\u09b0\u09be \u09aa\u09cd\u09b0\u09a4\u09bf\u0995\u09be\u09b0 \u0996\u09c1\u0981\u099c\u09c7 \u09aa\u09be\u09ac! &quot; module_class=\u201dsign-btn\u201d _builder_version=\u201d4.16\u2033 header_font_size=\u201d25px\u201d background_color=\u201d#29327a\u201d animation_style=\u201dslide\u201d animation_direction=\u201dleft\u201d animation_intensity_slide=\u201dT_font_slide=\u201dT_font_slide=\u201d251 header_font_size_phone=\u201d30px\u201d header_font_size_last_edited=\u201don|desktop\u201d body_font_size_tablet=\u201d\u201d body_font_size_phone=\u201d\u201d body_font_size_last_edited=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 \u09ac\u09b0\u09cd\u09a1\u09be\u09b0_\u09b0\u09be\u09a6\u09bf=\u201d\u0985\u09a8|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]=\u201d]<\/p>\n<h2>\u098f\u0995\u09b8\u09be\u09a5\u09c7, \u0986\u09ae\u09b0\u09be<\/h2>\n<h2><em>\u0989\u0987\u09b2<\/em><\/h2>\n<h2>\u09aa\u09cd\u09b0\u09a4\u09bf\u0995\u09be\u09b0 \u0996\u09c1\u0981\u099c\u09c7!<\/h2>\n<p>[\/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=\u201d{}\u201d 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title_font_size=&#8221;55&#8243; background_color=&#8221;#29327a&#8221; background_image=&#8221;https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg&#8221; background_position=&#8221;center_left&#8221; custom_padding=&#8221;9vw||9vw||true&#8221; custom_padding_tablet=&#8221;&#8221; custom_padding_phone=&#8221;|56px||&#8221; custom_padding_last_edited=&#8221;on|desktop&#8221; title_font_size_tablet=&#8221;45px&#8221; title_font_size_phone=&#8221;40px&#8221; title_font_size_last_edited=&#8221;on|phone&#8221; z_index_tablet=&#8221;500&#8243; custom_css_main_element=&#8221;background-position: center 18% !important;&#8221; global_colors_info=&#8221;{}&#8221;] Immortalized Cell Culture Protocols &nbsp; [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=&#8221;1&#8243; use_custom_gutter=&#8221;on&#8221; gutter_width=&#8221;1&#8243; specialty=&#8221;on&#8221; padding_left_1=&#8221;35px&#8221; padding_left_2=&#8221;35px&#8221; 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use_theme_fonts=\"yes\"][vc_column_text]\r\n<h2>Immortalized Cell Culture Protocol<\/h2>\r\nWe thank Dr Martin Dorf at Harvard University for generously generating and providing the\u00a0immortalized cell lines to the PRF Cell & Tissue Bank. Thank you.\r\n\r\nPRF cell lines were immortalized using the following protocol:\r\n<h3><strong>Retrovirus Infection<\/strong><\/h3>\r\n<strong>A. Producing Virus:<\/strong> 12-24 hr before transfection, packaging HEK293 cells were plated on a 100-mm plate at 60-80% confluency. Each plate was co-transfected with 12\u03bcg retroviral construct\u00a0pBABE-puro (SV40T) and 12\u03bcg pCL-Ampho Retrovirus Packaging Vector. The culture medium\u00a0was aspirated 8-10 hr after transfection, and replaced with 10ml of complete medium. The\u00a0culture was incubated for an additional 48-72 hr to attain optimal viral titer.\r\n\r\n<strong>B. Infecting Target Cells:<\/strong> The target cells were plated on a 100 mm plate 12-18 hr before\u00a0infection. The medium from packaging cells was collected and filtered through a 0.45-\u03bcm\u00a0cellulose acetate or polysulfonic filter. Once the cells were 40-60% confluent, 8 ml virus-containing medium with a final concentration of 10 \u03bcg\/ml polybrene was added. Three rounds\u00a0of infection were performed sequentially, ~12 hrs apart. The medium was replaced after 24 hr\u00a0incubation.\r\n\r\n<strong>C. Selection of Stable Cell Lines:<\/strong> 72 hr after infection, the cells were trypsinized, split at 1:3 and\u00a0transferred to two 100 mm plates in the presence of 3 \u03bcg\/ml puromycin, plus one control plate\u00a0without antibiotic. The culture medium was changed every 2 or 3 days for approximately 2 to 4\u00a0weeks to produce stable cell lines.\r\n<h2><b>Protocol for subculturing and freezing immortalized fibroblasts<\/b><b>\u00a0<\/b><\/h2>\r\n<table border=\"0\">\r\n<tbody>\r\n<tr>\r\n<td align=\"left\" valign=\"top\"><strong>Media<\/strong><\/td>\r\n<td align=\"left\" valign=\"top\"><strong>\u00a0 Trypsin<\/strong><\/td>\r\n<\/tr>\r\n<tr>\r\n<td align=\"left\" valign=\"top\">DMEM- Invitrogen #11960-044 (high glucose without L-glutamine)<\/td>\r\n<td align=\"left\" valign=\"top\">\u00a0 Trypsin EDTA C .25% (Invitrogen#25200-056)<\/td>\r\n<\/tr>\r\n<tr>\r\n<td align=\"left\" valign=\"top\">15% FBS Fetal Bovine Serum<\/td>\r\n<td align=\"left\" valign=\"top\"><b>\u00a0 Hank\u2019s Balanced Salt Solution<\/b><\/td>\r\n<\/tr>\r\n<tr>\r\n<td align=\"left\" valign=\"top\">1% (1x) Penicillin-Streptomycin (Invitrogen#15140-122)<\/td>\r\n<td align=\"left\" valign=\"top\">\u00a0 HBSS- (Invitrogen#14170 (1X)\u00a0 (-) calcium Chloride,\u00a0 (-)magnesium chloride,\u00a0 (-) magnesium sulfate)<\/td>\r\n<\/tr>\r\n<tr>\r\n<td align=\"left\" valign=\"top\">1% (1X) L-glutamine (Invitrogen#25030-081)<\/td>\r\n<td align=\"left\" valign=\"top\"><b>\u00a0 DMSO for Freezing<\/b><\/td>\r\n<\/tr>\r\n<tr>\r\n<td align=\"left\" valign=\"top\">0.75 \u03bcg\/ml puromycin (InvivoGen #ant-pr-1)<\/td>\r\n<td align=\"left\" valign=\"top\">\u00a0 Cell Culture Grade DMSO<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n<b>Cells will arrive frozen on dry ice at a concentration of approximately 5 x 10<sup>5<\/sup>cells\/vial<\/b>\r\n<h3><b>Protocol for thawing fibroblasts<\/b><\/h3>\r\n<ol>\r\n \t<li>Thaw cells rapidly in a 37\u00b0C water bath.<\/li>\r\n \t<li>Wipe the outside of the vial with 70% ethanol.<\/li>\r\n \t<li>Transfer the thawed cells to a T25 flask containing 5 ml of growth media without puromycin.<\/li>\r\n \t<li>Place flask in 37\u00b0C incubator overnight.<\/li>\r\n \t<li>The following day, observe under the microscope to ensure cells have attached.<\/li>\r\n \t<li>Remove media and replace with fresh growth media.<\/li>\r\n<\/ol>\r\n<h3><b>Subculturing PRF cell lines<\/b><\/h3>\r\n1) When cells are established and growing, begin to use media containing 0.75 \u03bcg\/ml puromycin. Continue to maintain the cells in media containing 0.75 \u03bcg\/ml puromycin.\r\n\r\n2) Cells should be split when confluent.\r\n\r\n3) To split cells (volumes given are assuming cells are in a T25):\r\n\r\nA. Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.\r\n\r\nB. 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. If cells are not detached after 3 minutes, you may incubate another 1-2 minutes\r\n\r\nC. 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\n\r\nD. 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 in to 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\n\r\nE. Using sterile technique, remove an aliquot for counting on a hemacytometer.\r\n\r\nF. While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpms.\r\n\r\n4) After calculating your cell count, plate cells at 2.5 x\u00a010<sup>5<\/sup>\u00a0cells per T 25 filter top flask.\r\n\r\n5) Cells should be fed every 2-3 days with fresh growth medium containing 0.75 \u03bcg\/ml puromycin.\r\n\r\n<b>Freezing:\r\n<\/b>Cells should be frozen at no less than 5x 10<sup>5<\/sup> cells \/ml\/cryovial\u00a0<span style=\"color: #000000;\">in growth media<\/span> containing 10% DMSO and 30% FBS <strong>without puromycin<\/strong> and subsequently placed in an isopropanol freezing chamber at -80\u00b0C overnight. Transfer to the liquid nitrogen the next day.\r\n<h4><b>For Further Information Please Contact:<\/b><\/h4>\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\r\nWarren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\r\nDepartment of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA\r\nMedical Director, The Progeria Research Foundation\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\n<a href=\"mailto:Leslie_Gordon@brown.edu\">Leslie_Gordon@brown.edu\u00a0<\/a><a href=\"https:\/\/www.progeriaresearch.org\/Leslie_Gordon@brown.edu\">\r\n<\/a>\r\n<div><span style=\"font-family: Georgia, Palatino;\">Wendy Norris<\/span><\/div>\r\n<div><span style=\"font-family: Georgia, Palatino;\">PRF Cell and Tissue Bank\r\nPhone: 401-274-1122 x 48063\r\n<a href=\"mailto:wnorris@lifespan.org\" target=\"_blank\" rel=\"noopener noreferrer\">wnorris@lifespan.org<\/a><\/span><\/div>\r\n[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1008","page","type-page","status-publish","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Immortalized | The Progeria Research Foundation<\/title>\n<meta name=\"description\" content=\"Dr Martin Dorf at Harvard University has generously generated and provided the immortalized cell lines to the PRF Cell &amp; Tissue Bank.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.progeriaresearch.org\/bn\/immortalized-cell-culture-protocols\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" 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