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\n[\/et_pb_sidebar][\/et_pb_column][et_pb_column type=\u201d3_4\u2033 specialty_columns=\u201d3\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_row_inner _builder_version=\u201d4.16\u2033 background_color=\u201d#00b2e2\u2033 custom_margin=\u201d41px||\u201d custom_margin_tablet=\u201d0px||\u201d custom_margin_phone=\u201d\u201d custom_margin_last_edited=\u201don|phone\u201d custom_padding=\u201d39.4375px|20px|0|20px|false|true\u201d animation_style=\u201dslide\u201d animation_direction=\u201dtop\u201d animation_intensity_slide=\u201d25%\u201d box_shadow_style=\u201dpreset1\u2033 box_shadow_blur=\u201d38px\u201d box_shadow_spread=\u201d-12px\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 admin_label=\u201dO trabalho da Progeria Research Foundation\u2026\u201d _builder_version=\u201d4.16\u2033 header_2_line_height=\u201d1.2em\u201d background_layout=\u201ddark\u201d custom_margin=\u201d||35px\u201d custom_padding=\u201d|||\u201d custom_padding_tablet=\u201d|50px||50px||true\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201dno|desktop\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d\u201d header_font_size_last_edited=\u201dno|desktop\u201d header_2_font_size_tablet=\u201d25px\u201d header_2_font_size_phone=\u201d\u201d header_2_font_size_last_edited=\u201dno|tablet\u201d header_4_font_size_tablet=\u201d\u201d header_4_font_size_phone=\u201d19px\u201d header_4_font_size_last_edited=\u201dno|telefone\u201d header_4_line_height_tablet=\u201d\u201d header_4_line_height_phone=\u201d1.2em\u201d header_4_line_height_last_edited=\u201dno|telefone\u201d z_index_tablet=\u201d500\u2033 informa\u00e7\u00f5es_de_cores_globais=\u201d{}\u201d]<\/p>\n
[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner preenchimento_personalizado_\u00faltima_edi\u00e7\u00e3o=\u201dem|telefone\u201d _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d||0px|||\u201d preenchimento_personalizado_tablet=\u201d|35px||35px||true\u201d preenchimento_personalizado_telefone=\u201d\u201d largura_da_borda_superior=\u201d10px\u201d cor_da_borda_superior=\u201d#8fd2ed\u201d cores_globais_info=\u201d{}\u201d][et_pb_column_inner tipo_de_coluna_especialidade_salva=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.16\u2033 background_size=\u201dinicial\u201d background_position=\u201dtop_left\u201d background_repeat=\u201drepetir\u201d custom_padding=\u201d||0px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n
Agradecemos ao Dr. Martin Dorf da Universidade de Harvard por generosamente gerar e fornecer as linhas celulares imortalizadas para o PRF Cell & Tissue Bank. Obrigado.<\/p>\n
As linhas celulares PRF foram imortalizadas usando o seguinte protocolo:<\/p>\n
A. Produ\u00e7\u00e3o de v\u00edrus:<\/strong> 12-24 h antes da transfec\u00e7\u00e3o, as c\u00e9lulas HEK293 de embalagem foram semeadas em uma placa de 100 mm na conflu\u00eancia de 60-80%. Cada placa foi cotransfetada com 12 \u03bcg de constru\u00e7\u00e3o retroviral pBABE-puro (SV40T) e 12 \u03bcg de vetor de embalagem de retrov\u00edrus pCL-Ampho. O meio de cultura foi aspirado 8-10 h ap\u00f3s a transfec\u00e7\u00e3o e substitu\u00eddo por 10 ml de meio completo. A cultura foi incubada por mais 48-72 h para atingir o t\u00edtulo viral ideal.<\/p>\n B. Infectando c\u00e9lulas-alvo:<\/strong> As c\u00e9lulas alvo foram plaqueadas em uma placa de 100 mm 12-18 horas antes da infec\u00e7\u00e3o. O meio das c\u00e9lulas de embalagem foi coletado e filtrado atrav\u00e9s de um filtro de acetato de celulose ou polissulf\u00f4nico de 0,45 \u03bcm. Uma vez que as c\u00e9lulas estavam confluentes 40-60%, 8 ml de meio contendo v\u00edrus com uma concentra\u00e7\u00e3o final de 10 \u03bcg\/ml de polibreno foram adicionados. Tr\u00eas rodadas de infec\u00e7\u00e3o foram realizadas sequencialmente, com ~12 horas de intervalo. O meio foi substitu\u00eddo ap\u00f3s 24 horas de incuba\u00e7\u00e3o.<\/p>\n C. Sele\u00e7\u00e3o de linhas celulares est\u00e1veis:<\/strong> 72 h ap\u00f3s a infec\u00e7\u00e3o, as c\u00e9lulas foram tripsinizadas, divididas em 1:3 e transferidas para duas placas de 100 mm na presen\u00e7a de 3 \u03bcg\/ml de puromicina, mais uma placa de controle sem antibi\u00f3tico. O meio de cultura foi trocado a cada 2 ou 3 dias por aproximadamente 2 a 4 semanas para produzir linhagens celulares est\u00e1veis.<\/p>\n <\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\u201dno|telefone\u201d _builder_version=\u201d4.16\u2033 custom_margin=\u201d|||||\u201d preenchimento_personalizado=\u201d0px|35px|0|0px|falso|falso\u201d preenchimento_personalizado_tablet=\u201d|35px|20px|35px||true\u201d preenchimento_personalizado_phone=\u201d|35px|11px|35px|false|true\u201d dire\u00e7\u00e3o_anima\u00e7\u00e3o=\u201dtopo\u201d informa\u00e7\u00f5es_de_cores_global=\u201d{}\u201d][inner_coluna_et_pb tipo=\u201d1_2\u2033 tipo_de_coluna_de_especialidade_salva=\u201d3_4\u2033 vers\u00e3o_do_construtor=\u201d4.16\u2033 preenchimento_personalizado=\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=\u201d4.16\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 DMEM- Invitrogen #11960-044 (alta glicose sem L-glutamina)<\/span><\/p>\n 15% FBS Soro Fetal Bovino<\/span><\/p>\n 1% (1x) Penicilina-Estreptomicina (Invitrogen#15140-122)<\/span><\/p>\n 1% (1X) L-glutamina (Invitrogen#25030-081)<\/span><\/p>\n 0,75 \u03bcg\/ml puromicina (InvivoGen #ant-pr-1)<\/span><\/p>\n [\/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 Tripsina EDTA C .25% (Invitrogen#25200-056)<\/span><\/p>\n HBSS- (Invitrogen#14170 (1X) (-) cloreto de c\u00e1lcio, (-) cloreto de magn\u00e9sio, (-) sulfato de magn\u00e9sio)<\/span><\/p>\n DMSO de grau de cultura celular<\/span><\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner preenchimento_personalizado_\u00faltima_edi\u00e7\u00e3o=\u201dno|telefone\u201d _builder_version=\u201d4.16\u2033 margem_personalizada=\u201d15px||35px|||\u201d preenchimento_personalizado=\u201d0px|35px|0|0px|falso|falso\u201d preenchimento_personalizado_tablet=\u201d|35px|20px|35px||true\u201d preenchimento_personalizado_phone=\u201d|35px|11px|35px|false|true\u201d dire\u00e7\u00e3o_anima\u00e7\u00e3o=\u201dtopo\u201d informa\u00e7\u00f5es_de_cores_globais=\u201d{}\u201d][inner_coluna_et_pb tipo_de_coluna_especialidade_salvo=\u201d3_4\u2033 _vers\u00e3o_do_construtor=\u201d4.16\u2033 preenchimento_personalizado=\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=\u201d4.16\u2033 custom_margin=\u201d||35px\u201d global_colors_info=\u201d{}\u201d]<\/p>\n As c\u00e9lulas chegar\u00e3o congeladas em gelo seco em uma concentra\u00e7\u00e3o de aproximadamente 5 x 105<\/sup>c\u00e9lulas\/frasco<\/b><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u201d4.16\u2033 background_size=\u201dinicial\u201d background_position=\u201dtopo_esquerdo\u201d background_repeat=\u201drepetir\u201d custom_padding=\u201d||0px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n 1) Quando as c\u00e9lulas estiverem estabelecidas e crescendo, comece a usar meios contendo 0,75 \u03bcg\/ml de puromicina. Continue a manter as c\u00e9lulas em meios contendo 0,75 \u03bcg\/ml de puromicina.<\/p>\n 2) As c\u00e9lulas devem ser divididas quando confluentes.<\/p>\n 3) Para dividir c\u00e9lulas (os volumes fornecidos pressup\u00f5em que as c\u00e9lulas estejam em um T25):<\/p>\n A. Usando t\u00e9cnica est\u00e9ril, remova o meio e lave o frasco com 2-3 ml de HBSS est\u00e9ril. Remova e descarte o HBSS.<\/p>\n B. Adicione 1 ml de tripsina e incubar por 2-3 minutos. As c\u00e9lulas devem ser verificadas sob um microsc\u00f3pio invertido para determinar se elas come\u00e7aram a se arredondar, levantar e flutuar. Se as c\u00e9lulas n\u00e3o se destacarem ap\u00f3s 3 minutos, voc\u00ea pode incubar por mais 1-2 minutos<\/p>\n C. Aperte a tampa e incline suavemente o frasco de um lado para o outro para desalojar todas as c\u00e9lulas. O frasco pode ser batido levemente na lateral para soltar as c\u00e9lulas, se necess\u00e1rio.<\/p>\n D. Adicione 4 ml de novo meio ao frasco assim que as c\u00e9lulas estiverem flutuando para inativar a tripsina. Enx\u00e1gue as laterais do frasco v\u00e1rias vezes com o novo meio para lavar todas as c\u00e9lulas do pl\u00e1stico e para dentro da solu\u00e7\u00e3o. Remova todo o l\u00edquido contendo c\u00e9lulas e transfira para um recipiente c\u00f4nico de 15 ml (ou 50 ml se voc\u00ea estiver reunindo 3 ou mais frascos).<\/p>\n E. Usando t\u00e9cnica est\u00e9ril, remova uma al\u00edquota para contagem em um hemacit\u00f4metro.<\/p>\n F. Enquanto voc\u00ea conta as c\u00e9lulas, o restante da solu\u00e7\u00e3o deve ser centrifugado na centr\u00edfuga cl\u00ednica por 5 minutos a 1000 rpm.<\/p>\n 4) Ap\u00f3s calcular a contagem de c\u00e9lulas, separe as c\u00e9lulas em 2,5 x 105<\/sup>\u00a0c\u00e9lulas por frasco com filtro T 25.<\/p>\n 5) As c\u00e9lulas devem ser alimentadas a cada 2-3 dias com meio de crescimento fresco contendo 0,75 \u03bcg\/ml de puromicina.<\/p>\n <\/b>As c\u00e9lulas devem ser congeladas a uma temperatura n\u00e3o inferior a 5x105<\/sup>\u00a0<\/span>c\u00e9lulas \/ml\/criotubo\u00a0em meios de crescimento<\/span>\u00a0<\/span>contendo 10% DMSO e 30% FBS\u00a0<\/span>sem puromicina<\/strong>\u00a0<\/span>e posteriormente colocada em uma c\u00e2mara de congelamento de isopropanol a -80\u00b0C durante a noite. Transfira para o nitrog\u00eanio l\u00edquido no dia seguinte.<\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner preenchimento_personalizado_\u00faltima_edi\u00e7\u00e3o=\u201dem|telefone\u201d _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d||0px|||\u201d preenchimento_personalizado_tablet=\u201d|35px||35px||true\u201d preenchimento_personalizado_telefone=\u201d\u201d largura_da_borda_superior=\u201d10px\u201d cor_da_borda_superior=\u201d#8fd2ed\u201d bloqueado=\u201ddesligado\u201d cores_globais_info=\u201d{}\u201d][et_pb_column_inner tipo_de_coluna_especialidade_salva=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d|||\u201d global_colors_info=\u201d{}\u201d preenchimento_personalizado__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.16\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\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 Professor de Pesquisa Pedi\u00e1trica Warren Alpert Medical School da Brown University e Departamento de Pediatria, Hasbro Children's Hospital, Providence, RI Departamento de Anestesia, Children's Hospital Boston e Harvard Medical School, Boston, MA Diretor M\u00e9dico, The Progeria Research Foundation<\/p>\n Telefone: 978-535-2594 [\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner tipo=\u201d1_2\u2033 tipo_de_coluna_de_especialidade_salva=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d|||20px\u201d preenchimento_personalizado_tablet=\u201d20px|||0px\u201d preenchimento_personalizado_phone=\u201d\u201d preenchimento_personalizado_last_edited=\u201dem|telefone\u201d global_colors_info=\u201d{}\u201d preenchimento_personalizado_hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 preenchimento_personalizado=\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 [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 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bot\u00e3o_border_color__hover_enabled=\u201don\u201d]<\/p>\n [\/et_pb_cta][\/et_pb_column][et_pb_column tipo=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 preenchimento_personalizado=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_image src=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2024\/08\/2024-strip-footer-strip-copy.png\u201d title_text=\u201d2024 c\u00f3pia de tira de rodap\u00e9\u201d _builder_version=\u201d4.27.0\u2033 _module_preset=\u201ddefault\u201d custom_margin=\u201d35px||||false|false\u201d global_colors_info=\u201d{}\u201d][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>","protected":false},"excerpt":{"rendered":" [et_pb_section fb_built=\u201d1\u2033 largura_completa=\u201dligado\u201d disabled_on=\u201ddesligado|desligado|desligado\u201d _builder_version=\u201d4.16\u2033 largura_completa_bottom=\u201d55px\u201d cor_completa_bottom=\u201d#29327a\u201d bloqueado=\u201ddesligado\u201d global_colors_info=\u201d{}\u201d][et_pb_cabe\u00e7alho_completo _builder_version=\u201d4.16\u2033 fonte_completa=\u201d||||||||\u201d title_font_size=\u201d55\u2033 background_color=\u201d#29327a\u201d background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg\u201d background_position=\u201dcenter_left\u201d custom_padding=\u201d9vw||9vw||true\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201don|desktop\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201don|phone\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dposi\u00e7\u00e3o de fundo: centro 18% !importante;\u201d global_colors_info=\u201d{}\u201d] Protocolos de cultura de c\u00e9lulas imortalizadas [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 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[\u2026]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Immortalized Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\nM\u00eddia de crescimento<\/h4>\n
Tripsina<\/strong><\/h4>\n
Solu\u00e7\u00e3o salina balanceada de Hank<\/strong><\/h4>\n
DMSO para Congelamento<\/strong><\/h4>\n
Protocolo para descongelamento de fibroblastos<\/b><\/h4>\n
\n
Subcultura de linhas celulares PRF<\/b><\/h4>\n
Congelamento:<\/h4>\n
Para mais informa\u00e7\u00f5es, entre em contato com:<\/strong><\/h4>\n
Dr. Leslie B. Gordon, PhD<\/h6>\n
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\nWendy Norris<\/span><\/h6>\n
Telefone: 401-274-1122 x 48063
wnorris@brownhealth.org<\/a><\/a><\/span><\/div>\nInscrever-se<\/h2>\n
para o nosso<\/h2>\n
Boletim informativo!<\/h2>\n
Juntos, n\u00f3s<\/h2>\n
VAI<\/em><\/h2>\n
encontre a cura!<\/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