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[\/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” use_custom_width=”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_sidebar area=”et_pb_widget_area_14″ disabled_on=”on|on|off” module_class=”subpage-sidebars” _builder_version=”4.16″ animation_style=”fade” z_index_tablet=”500″ border_width_right=”5px” locked=”off” global_colors_info=”{}”]
\n[\/et_pb_sidebar][\/et_pb_column][et_pb_column type=”3_4″ specialty_columns=”3″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_row_inner _builder_version=”4.16″ background_color=”#00b2e2″ custom_margin=”41px||” custom_margin_tablet=”0px||” custom_margin_phone=”” custom_margin_last_edited=”on|phone” custom_padding=”39.4375px|20px|0|20px|false|true” animation_style=”slide” animation_direction=”top” animation_intensity_slide=”25%” box_shadow_style=”preset1″ box_shadow_blur=”38px” box_shadow_spread=”-12px” 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 admin_label=”The Progeria Research Foundation\u2019s work…” _builder_version=”4.16″ header_2_line_height=”1.2em” background_layout=”dark” custom_margin=”||35px” custom_padding=”|||” custom_padding_tablet=”|50px||50px||true” custom_padding_phone=”” custom_padding_last_edited=”on|desktop” header_font_size_tablet=”” header_font_size_phone=”” header_font_size_last_edited=”on|desktop” header_2_font_size_tablet=”25px” header_2_font_size_phone=”” header_2_font_size_last_edited=”on|tablet” header_4_font_size_tablet=”” header_4_font_size_phone=”19px” header_4_font_size_last_edited=”on|phone” header_4_line_height_tablet=”” header_4_line_height_phone=”1.2em” header_4_line_height_last_edited=”on|phone” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\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” 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″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||0px|||” global_colors_info=”{}”]<\/p>\n
We thank Dr Martin Dorf at Harvard University for generously generating and providing the\u00a0immortalized cell lines to the PRF Cell & Tissue Bank. Thank you.<\/p>\n
PRF cell lines were immortalized using the following protocol:<\/p>\n
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.<\/p>\n 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.<\/p>\n 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.<\/p>\n <\/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_margin=”|||||” custom_padding=”0px|35px|0|0px|false|false” custom_padding_tablet=”|35px|20px|35px||true” custom_padding_phone=”|35px|11px|35px|false|true” animation_direction=”top” 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=”4.16″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||24px|||” global_colors_info=”{}”]<\/p>\n DMEM- Invitrogen #11960-044 (high glucose without L-glutamine)<\/span><\/p>\n 15% FBS Fetal Bovine Serum<\/span><\/p>\n 1% (1x) Penicillin-Streptomycin (Invitrogen#15140-122)<\/span><\/p>\n 1% (1X) L-glutamine (Invitrogen#25030-081)<\/span><\/p>\n 0.75 \u03bcg\/ml puromycin (InvivoGen #ant-pr-1)<\/span><\/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=”4.16″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||24px|||” global_colors_info=”{}”]<\/p>\n Trypsin EDTA C .25% (Invitrogen#25200-056)<\/span><\/p>\n HBSS- (Invitrogen#14170 (1X)\u00a0 (-) calcium Chloride,\u00a0 (-)magnesium chloride,\u00a0 (-) magnesium sulfate)<\/span><\/p>\n Cell Culture Grade DMSO<\/span><\/p>\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_margin=”15px||35px|||” custom_padding=”0px|35px|0|0px|false|false” custom_padding_tablet=”|35px|20px|35px||true” custom_padding_phone=”|35px|11px|35px|false|true” animation_direction=”top” global_colors_info=”{}”][et_pb_column_inner 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=”4.16″ custom_margin=”||35px” global_colors_info=”{}”]<\/p>\n Cells will arrive frozen on dry ice at a concentration of approximately 5 x 105<\/sup>cells\/vial<\/b><\/p>\n [\/et_pb_text][et_pb_text _builder_version=”4.16″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||0px|||” global_colors_info=”{}”]<\/p>\n 1) 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.<\/p>\n 2) Cells should be split when confluent.<\/p>\n 3) To split cells (volumes given are assuming cells are in a T25):<\/p>\n A. Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.<\/p>\n B. 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<\/p>\n C. 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.<\/p>\n D. 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).<\/p>\n E. Using sterile technique, remove an aliquot for counting on a hemacytometer.<\/p>\n F. While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpms.<\/p>\n 4) After calculating your cell count, plate cells at 2.5 x\u00a0105<\/sup>\u00a0cells per T 25 filter top flask.<\/p>\n 5) Cells should be fed every 2-3 days with fresh growth medium containing 0.75 \u03bcg\/ml puromycin.<\/p>\n <\/b>Cells should be frozen at no less than 5x 105<\/sup>\u00a0<\/span>cells \/ml\/cryovial\u00a0in growth media<\/span>\u00a0<\/span>containing 10% DMSO and 30% FBS\u00a0<\/span>without puromycin<\/strong>\u00a0<\/span>and subsequently placed in an isopropanol freezing chamber at -80\u00b0C overnight. Transfer to the liquid nitrogen the next day.<\/p>\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″ custom_padding=”||0px|||” 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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=”{}”] Immortalized 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=\"Immortalized Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\nGrowth Media<\/h4>\n
Trypsin<\/strong><\/h4>\n
Hank\u2019s Balanced Salt Solution<\/strong><\/h4>\n
DMSO for Freezing<\/strong><\/h4>\n
Protocol for thawing fibroblasts<\/b><\/h4>\n
\n
Subculturing PRF cell lines<\/b><\/h4>\n
Freezing:<\/h4>\n
For 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><\/a><\/span><\/div>\nSign Up<\/h2>\n
for Our<\/h2>\n
Newsletter!<\/h2>\n
Together, we<\/h2>\n
WILL<\/em><\/h2>\n
find the cure!<\/h2>\n
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
Retrovirus Infection<\/strong><\/h3>\r\nA. 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\nB. 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\nC. 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
Protocol for subculturing and freezing immortalized fibroblasts<\/b>\u00a0<\/b><\/h2>\r\n
\r\n\r\n
\r\n Media<\/strong><\/td>\r\n \u00a0 Trypsin<\/strong><\/td>\r\n<\/tr>\r\n \r\n DMEM- Invitrogen #11960-044 (high glucose without L-glutamine)<\/td>\r\n \u00a0 Trypsin EDTA C .25% (Invitrogen#25200-056)<\/td>\r\n<\/tr>\r\n \r\n 15% FBS Fetal Bovine Serum<\/td>\r\n \u00a0 Hank\u2019s Balanced Salt Solution<\/b><\/td>\r\n<\/tr>\r\n \r\n 1% (1x) Penicillin-Streptomycin (Invitrogen#15140-122)<\/td>\r\n \u00a0 HBSS- (Invitrogen#14170 (1X)\u00a0 (-) calcium Chloride,\u00a0 (-)magnesium chloride,\u00a0 (-) magnesium sulfate)<\/td>\r\n<\/tr>\r\n \r\n 1% (1X) L-glutamine (Invitrogen#25030-081)<\/td>\r\n \u00a0 DMSO for Freezing<\/b><\/td>\r\n<\/tr>\r\n \r\n 0.75 \u03bcg\/ml puromycin (InvivoGen #ant-pr-1)<\/td>\r\n \u00a0 Cell Culture Grade DMSO<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\nCells will arrive frozen on dry ice at a concentration of approximately 5 x 105<\/sup>cells\/vial<\/b>\r\n Protocol for thawing fibroblasts<\/b><\/h3>\r\n
\r\n \t
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\u00a0105<\/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\nFreezing:\r\n<\/b>Cells should be frozen at no less than 5x 105<\/sup> cells \/ml\/cryovial\u00a0in growth media<\/span> containing 10% DMSO and 30% FBS 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
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\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n<\/a>\r\n