Thawing PRF cell lines<\/strong><\/h4>\n\n- Thaw cells rapidly in a 37\u02daC water bath.<\/li>\n
- Wipe the outside of the vial with 70% ethanol.<\/li>\n
- Transfer the thawed cells to a T25 flask containing 5 ml of growth media.<\/li>\n
- Place flask in 37\u02daC incubator overnight.<\/li>\n
- The following day, observe under the microscope to ensure cells have attached.<\/li>\n
- Remove media and replace with fresh growth media.<\/li>\n<\/ol>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n\n\n\n\n\n\n
Subculturing PRF cell lines<\/strong><\/h4>\n\n- Cells should be split when confluent.<\/li>\n
- To split cells (volumes given are assuming cells are in a T25):
a)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.
b)\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.
c)\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.
d)\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).
e)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.
f)\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>\n - After calculating your cell count, plate cells at 2.5 x 105<\/sup>\u00a0<\/span>cells per T25 filter top flask.<\/li>\n
- Cells should be fed every 2-3 days with fresh growth medium.<\/li>\n<\/ol>\n
Freezing:
<\/u><\/strong><\/h4>\nCells should be frozen at no less than 5 x 105<\/sup>\u00a0<\/span>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.<\/p>\nProtocol for Subculturing and Freezing Fibroblasts<\/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=”on|phone” _builder_version=”4.16″ custom_padding=”||0px|||” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” border_width_top=”10px” border_color_top=”#8fd2ed” locked=”off” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.16″ global_colors_info=”{}”]<\/p>\n
For Further Information Please Contact:<\/strong><\/h4>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=”1_2,1_2″ custom_padding_last_edited=”on|phone” _builder_version=”4.16″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” locked=”off” global_colors_info=”{}”][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|20px||” 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″ global_colors_info=”{}”]<\/p>\n
Leslie B. Gordon, MD, PhD<\/h6>\n
Professor 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<\/p>\n
Phone: 978-535-2594
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||20px” custom_padding_tablet=”20px|||0px” custom_padding_phone=”” custom_padding_last_edited=”on|phone” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.2″ custom_padding=”||0px|||” custom_padding_last_edited=”off|desktop” hover_enabled=”0″ global_colors_info=”{}” sticky_enabled=”0″]<\/p>\n
Wendy Norris<\/span><\/h6>\nPRF Cell and Tissue Bank
Phone: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
<\/span><\/div>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=”1″ module_class=”footer” _builder_version=”4.21.0″ background_color=”#29327a” custom_margin=”-2px|||||” custom_padding=”0|0px|0|0px|false|false” z_index_tablet=”500″ border_width_top=”12px” border_color_top=”#00b2e2″ global_module=”133″ locked=”off” global_colors_info=”{}”][et_pb_row_inner column_structure=”1_3,1_3,1_3″ disabled_on=”on|on|on” admin_label=”Row” _builder_version=”4.21.0″ _module_preset=”default” min_height=”277px” custom_margin=”|20px|30px||false|false” custom_padding=”0px|30px|0px||false|false” disabled=”on” global_colors_info=”{}”][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” 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\/” align=”right” _builder_version=”4.21.0″ _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″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” global_colors_info=”{}”][\/et_pb_column_inner][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _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_version=”4.21.0″ _module_preset=”default” custom_margin=”|20px||-50px|false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row column_structure=”1_4,1_4,1_2″ make_equal=”on” module_class=” et_pb_row_fullwidth” _builder_version=”4.16″ width=”89%” width_tablet=”80%” width_phone=”” width_last_edited=”on|desktop” max_width=”89%” max_width_tablet=”80%” max_width_phone=”” max_width_last_edited=”on|desktop” z_index_tablet=”500″ make_fullwidth=”on” width_unit=”off” custom_width_percent=”100%” global_colors_info=”{}”][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_cta button_url=”https:\/\/www.progeriaresearch.org\/newsletter-signup\/” button_text=”Sign Up Now” admin_label=”Sign Up for Newsletter” module_class=”sign-btn” _builder_version=”4.16″ header_font_size=”25px” background_color=”#29327a” animation_style=”slide” animation_direction=”left” animation_intensity_slide=”25%” header_font_size_tablet=”” header_font_size_phone=”30px” header_font_size_last_edited=”on|desktop” z_index_tablet=”500″ border_radii=”on|25px|25px|25px|25px” global_colors_info=”{}” button_bg_color__hover_enabled=”on” button_bg_color__hover=”#8fd2ed” button_border_color__hover_enabled=”on”]<\/p>\n
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Together, we<\/h2>\nWILL<\/em><\/h2>\nfind the cure!<\/h2>\n
[\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” 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” title_text=”2024 strip footer strip copy” _builder_version=”4.27.0″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>\n","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>\n","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
Subculturing PRF cell lines<\/strong><\/h4>\n\n- Cells should be split when confluent.<\/li>\n
- To split cells (volumes given are assuming cells are in a T25):
a)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.
b)\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.
c)\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.
d)\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).
e)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.
f)\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>\n - After calculating your cell count, plate cells at 2.5 x 105<\/sup>\u00a0<\/span>cells per T25 filter top flask.<\/li>\n
- Cells should be fed every 2-3 days with fresh growth medium.<\/li>\n<\/ol>\n
Freezing:
<\/u><\/strong><\/h4>\nCells should be frozen at no less than 5 x 105<\/sup>\u00a0<\/span>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.<\/p>\nProtocol for Subculturing and Freezing Fibroblasts<\/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=”on|phone” _builder_version=”4.16″ custom_padding=”||0px|||” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” border_width_top=”10px” border_color_top=”#8fd2ed” locked=”off” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.16″ global_colors_info=”{}”]<\/p>\n
For Further Information Please Contact:<\/strong><\/h4>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=”1_2,1_2″ custom_padding_last_edited=”on|phone” _builder_version=”4.16″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” locked=”off” global_colors_info=”{}”][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|20px||” 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″ global_colors_info=”{}”]<\/p>\n
Leslie B. Gordon, MD, PhD<\/h6>\n
Professor 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<\/p>\n
Phone: 978-535-2594
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||20px” custom_padding_tablet=”20px|||0px” custom_padding_phone=”” custom_padding_last_edited=”on|phone” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.2″ custom_padding=”||0px|||” custom_padding_last_edited=”off|desktop” hover_enabled=”0″ global_colors_info=”{}” sticky_enabled=”0″]<\/p>\n
Wendy Norris<\/span><\/h6>\nPRF Cell and Tissue Bank
Phone: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
<\/span><\/div>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=”1″ module_class=”footer” _builder_version=”4.21.0″ background_color=”#29327a” custom_margin=”-2px|||||” custom_padding=”0|0px|0|0px|false|false” z_index_tablet=”500″ border_width_top=”12px” border_color_top=”#00b2e2″ global_module=”133″ locked=”off” global_colors_info=”{}”][et_pb_row_inner column_structure=”1_3,1_3,1_3″ disabled_on=”on|on|on” admin_label=”Row” _builder_version=”4.21.0″ _module_preset=”default” min_height=”277px” custom_margin=”|20px|30px||false|false” custom_padding=”0px|30px|0px||false|false” disabled=”on” global_colors_info=”{}”][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” 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\/” align=”right” _builder_version=”4.21.0″ _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″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” global_colors_info=”{}”][\/et_pb_column_inner][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _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_version=”4.21.0″ _module_preset=”default” custom_margin=”|20px||-50px|false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row column_structure=”1_4,1_4,1_2″ make_equal=”on” module_class=” et_pb_row_fullwidth” _builder_version=”4.16″ width=”89%” width_tablet=”80%” width_phone=”” width_last_edited=”on|desktop” max_width=”89%” max_width_tablet=”80%” max_width_phone=”” max_width_last_edited=”on|desktop” z_index_tablet=”500″ make_fullwidth=”on” width_unit=”off” custom_width_percent=”100%” global_colors_info=”{}”][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_cta button_url=”https:\/\/www.progeriaresearch.org\/newsletter-signup\/” button_text=”Sign Up Now” admin_label=”Sign Up for Newsletter” module_class=”sign-btn” _builder_version=”4.16″ header_font_size=”25px” background_color=”#29327a” animation_style=”slide” animation_direction=”left” animation_intensity_slide=”25%” header_font_size_tablet=”” header_font_size_phone=”30px” header_font_size_last_edited=”on|desktop” z_index_tablet=”500″ border_radii=”on|25px|25px|25px|25px” global_colors_info=”{}” button_bg_color__hover_enabled=”on” button_bg_color__hover=”#8fd2ed” button_border_color__hover_enabled=”on”]<\/p>\n
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Together, we<\/h2>\nWILL<\/em><\/h2>\nfind the cure!<\/h2>\n
[\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” 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” title_text=”2024 strip footer strip copy” _builder_version=”4.27.0″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>\n","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>\n","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
a)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.
b)\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.
c)\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.
d)\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).
e)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.
f)\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>\n
Freezing:
<\/u><\/strong><\/h4>\nCells should be frozen at no less than 5 x 105<\/sup>\u00a0<\/span>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.<\/p>\nProtocol for Subculturing and Freezing Fibroblasts<\/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=”on|phone” _builder_version=”4.16″ custom_padding=”||0px|||” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” border_width_top=”10px” border_color_top=”#8fd2ed” locked=”off” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.16″ global_colors_info=”{}”]<\/p>\n
For Further Information Please Contact:<\/strong><\/h4>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=”1_2,1_2″ custom_padding_last_edited=”on|phone” _builder_version=”4.16″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” locked=”off” global_colors_info=”{}”][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|20px||” 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″ global_colors_info=”{}”]<\/p>\n
Leslie B. Gordon, MD, PhD<\/h6>\n
Professor 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<\/p>\n
Phone: 978-535-2594
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\n[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||20px” custom_padding_tablet=”20px|||0px” custom_padding_phone=”” custom_padding_last_edited=”on|phone” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.2″ custom_padding=”||0px|||” custom_padding_last_edited=”off|desktop” hover_enabled=”0″ global_colors_info=”{}” sticky_enabled=”0″]<\/p>\n
Wendy Norris<\/span><\/h6>\nPRF Cell and Tissue Bank
Phone: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
<\/span><\/div>\n[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=”1″ module_class=”footer” _builder_version=”4.21.0″ background_color=”#29327a” custom_margin=”-2px|||||” custom_padding=”0|0px|0|0px|false|false” z_index_tablet=”500″ border_width_top=”12px” border_color_top=”#00b2e2″ global_module=”133″ locked=”off” global_colors_info=”{}”][et_pb_row_inner column_structure=”1_3,1_3,1_3″ disabled_on=”on|on|on” admin_label=”Row” _builder_version=”4.21.0″ _module_preset=”default” min_height=”277px” custom_margin=”|20px|30px||false|false” custom_padding=”0px|30px|0px||false|false” disabled=”on” global_colors_info=”{}”][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” 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\/” align=”right” _builder_version=”4.21.0″ _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″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _module_preset=”default” global_colors_info=”{}”][\/et_pb_column_inner][et_pb_column_inner type=”1_3″ saved_specialty_column_type=”3_4″ _builder_version=”4.21.0″ _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_version=”4.21.0″ _module_preset=”default” custom_margin=”|20px||-50px|false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row column_structure=”1_4,1_4,1_2″ make_equal=”on” module_class=” et_pb_row_fullwidth” _builder_version=”4.16″ width=”89%” width_tablet=”80%” width_phone=”” width_last_edited=”on|desktop” max_width=”89%” max_width_tablet=”80%” max_width_phone=”” max_width_last_edited=”on|desktop” z_index_tablet=”500″ make_fullwidth=”on” width_unit=”off” custom_width_percent=”100%” global_colors_info=”{}”][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_cta button_url=”https:\/\/www.progeriaresearch.org\/newsletter-signup\/” button_text=”Sign Up Now” admin_label=”Sign Up for Newsletter” module_class=”sign-btn” _builder_version=”4.16″ header_font_size=”25px” background_color=”#29327a” animation_style=”slide” animation_direction=”left” animation_intensity_slide=”25%” header_font_size_tablet=”” header_font_size_phone=”30px” header_font_size_last_edited=”on|desktop” z_index_tablet=”500″ border_radii=”on|25px|25px|25px|25px” global_colors_info=”{}” button_bg_color__hover_enabled=”on” button_bg_color__hover=”#8fd2ed” button_border_color__hover_enabled=”on”]<\/p>\n
Sign Up<\/h2>\nfor Our<\/h2>\nNewsletter!<\/h2>\n
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Together, we<\/h2>\nWILL<\/em><\/h2>\nfind the cure!<\/h2>\n
[\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” 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” title_text=”2024 strip footer strip copy” _builder_version=”4.27.0″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>\n","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>\n","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
Protocol for Subculturing and Freezing Fibroblasts<\/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=”on|phone” _builder_version=”4.16″ custom_padding=”||0px|||” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” border_width_top=”10px” border_color_top=”#8fd2ed” locked=”off” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.16″ global_colors_info=”{}”]<\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=”1_2,1_2″ custom_padding_last_edited=”on|phone” _builder_version=”4.16″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” locked=”off” global_colors_info=”{}”][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|20px||” 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″ global_colors_info=”{}”]<\/p>\n Professor 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<\/p>\n Phone: 978-535-2594 [\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=”1_2″ saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||20px” custom_padding_tablet=”20px|||0px” custom_padding_phone=”” custom_padding_last_edited=”on|phone” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.2″ 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button_bg_color__hover=”#8fd2ed” button_border_color__hover_enabled=”on”]<\/p>\n [\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_cta button_url=”https:\/\/progeriaresearch.donorsupport.co\/-\/XZHJVWZR” button_text=”Donate Now” admin_label=”Together, we will find the cure!” module_class=”sign-btn” _builder_version=”4.16″ header_font_size=”25px” background_color=”#29327a” animation_style=”slide” animation_direction=”left” animation_intensity_slide=”25%” header_font_size_tablet=”” 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″ border_radii=”on|25px|25px|25px|25px” global_colors_info=”{}” button_bg_color__hover_enabled=”on” button_bg_color__hover=”#8fd2ed” button_border_color__hover_enabled=”on”]<\/p>\n [\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” 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” title_text=”2024 strip footer strip copy” _builder_version=”4.27.0″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>\n","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>\n","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\nFor Further Information Please Contact:<\/strong><\/h4>\n
Leslie B. Gordon, MD, PhD<\/h6>\n
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\nWendy Norris<\/span><\/h6>\n
Phone: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a>
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WILL<\/em><\/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