Italiano: [et_pb_section fb_built=\u201d1\u2033 fullwidth=\u201don\u201d disabled_on=\u201doff|off|off\u201d _builder_version=\u201d4.16\u2033 border_width_bottom=\u201d55px\u201d border_color_bottom=\u201d#29327a\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 title_font=\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=\u201dbackground-position: center 18% !important;\u201d global_colors_info=\u201d{}\u201d]<\/p>\n
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[\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 use_custom_gutter=\u201don\u201d gutter_width=\u201d1\u2033 specialty=\u201don\u201d padding_left_1=\u201d35px\u201d padding_left_2=\u201d35px\u201d padding_2_tablet=\u201d|||0px\u201d padding_2_phone=\u201d\u201d padding_2_last_edited=\u201don|desktop\u201d module_class_1=\u201dsidebar-secondary-nav\u201d module_class=\u201dhandprint-bg\u201d _builder_version=\u201d4.16\u2033 background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png\u201d parallax=\u201don\u201d parallax_method=\u201doff\u201d larghezza_interna=\u201d100%\u201d larghezza_interna_massima=\u201d100%\u201d imbottitura_personalizzata=\u201d0|0px|54px|0px|falso|falso\u201d z_index_tablet=\u201d500\u2033 larghezza_interna_massima=\u201d10px\u201d colore_interna_massima=\u201d#8fd2ed\u201d usa_larghezza_personalizzata=\u201don\u201d unit\u00e0_di_larghezza=\u201doff\u201d percentuale_larghezza_personalizzata=\u201d100%\u201d info_colori_globali=\u201d{}\u201d][et_pb_column tipo=\u201d1_4\u2033 versione_builder=\u201d4.16\u2033 imbottitura_personalizzata=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_sidebar area=\u201det_pb_widget_area_14\u2033 disabled_on=\u201don|on|off\u201d module_class=\u201dsubpage-sidebars\u201d _builder_version=\u201d4.16\u2033 animation_style=\u201dfade\u201d z_index_tablet=\u201d500\u2033 border_width_right=\u201d5px\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d]
\nItaliano: [\/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=\u201dIl lavoro della 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=\u201don|desktop\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d\u201d header_font_size_last_edited=\u201don|desktop\u201d header_2_font_size_tablet=\u201d25px\u201d header_2_font_size_phone=\u201d\u201d header_2_font_size_last_edited=\u201don|tablet\u201d header_4_font_size_tablet=\u201d\u201d header_4_font_size_phone=\u201d19px\u201d header_4_font_size_last_edited=\u201don|phone\u201d header_4_line_height_tablet=\u201d\u201d header_4_line_height_phone=\u201d1.2em\u201d header_4_line_height_last_edited=\u201don|phone\u201d z_index_tablet=\u201d500\u2033 informazioni_globali_colori=\u201d{}\u201d]<\/p>\n
Italiano: [\/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 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 background_size=\u201dinitial\u201d background_position=\u201dtop_left\u201d background_repeat=\u201dripeti\u201d custom_padding=\u201d||0px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n
Ringraziamo il Dott. Martin Dorf dell'Universit\u00e0 di Harvard per aver generosamente generato e fornito le linee cellulari immortalizzate alla PRF Cell & Tissue Bank. Grazie.<\/p>\n
Le linee cellulari PRF sono state immortalate utilizzando il seguente protocollo:<\/p>\n
A. Produzione del virus:<\/strong> 12-24 ore prima della trasfezione, le cellule HEK293 di confezionamento sono state piastrate su una piastra da 100 mm a confluenza 60-80%. Ogni piastra \u00e8 stata co-trasfettata con 12\u03bcg di costrutto retrovirale pBABE-puro (SV40T) e 12\u03bcg di vettore di confezionamento retrovirus pCL-Ampho. Il terreno di coltura \u00e8 stato aspirato 8-10 ore dopo la trasfezione e sostituito con 10 ml di terreno completo. La coltura \u00e8 stata incubata per altre 48-72 ore per ottenere un titolo virale ottimale.<\/p>\n B. Infezione delle cellule bersaglio:<\/strong> Le cellule bersaglio sono state piastrate su una piastra da 100 mm 12-18 ore prima dell'infezione. Il terreno di coltura delle cellule di confezionamento \u00e8 stato raccolto e filtrato attraverso un filtro in acetato di cellulosa o polisulfonico da 0,45 \u03bcm. Una volta che le cellule erano 40-60% confluenti, sono stati aggiunti 8 ml di terreno contenente virus con una concentrazione finale di 10 \u03bcg\/ml di polibrene. Sono stati eseguiti tre cicli di infezione in sequenza, a distanza di circa 12 ore. Il terreno \u00e8 stato sostituito dopo 24 ore di incubazione.<\/p>\n C. Selezione di linee cellulari stabili:<\/strong> 72 ore dopo l'infezione, le cellule sono state tripsinizzate, divise a 1:3 e trasferite su due piastre da 100 mm in presenza di 3 \u03bcg\/ml di puromicina, pi\u00f9 una piastra di controllo senza antibiotico. Il terreno di coltura \u00e8 stato cambiato ogni 2 o 3 giorni per circa 2-4 settimane per produrre linee cellulari stabili.<\/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=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_margin=\u201d|||||\u201d custom_padding=\u201d0px|35px|0|0px|false|false\u201d custom_padding_tablet=\u201d|35px|20px|35px||true\u201d custom_padding_phone=\u201d|35px|11px|35px|false|true\u201d animation_direction=\u201dtop\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=\u201d4.16\u2033 background_size=\u201dinitial\u201d background_position=\u201dtop_left\u201d background_repeat=\u201dripeti\u201d custom_padding=\u201d||24px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n DMEM- Invitrogen #11960-044 (glucosio elevato senza L-glutammina)<\/span><\/p>\n 15% FBS siero fetale bovino<\/span><\/p>\n 1% (1x) Penicillina-Streptomicina (Invitrogen#15140-122)<\/span><\/p>\n 1% (1X) L-glutammina (Invitrogen#25030-081)<\/span><\/p>\n 0,75 \u03bcg\/ml di 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=\u201dripeti\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) (-) cloruro di calcio, (-) cloruro di magnesio, (-) solfato di magnesio)<\/span><\/p>\n DMSO di grado di coltura cellulare<\/span><\/p>\n Italiano: [\/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_margin=\u201d15px||35px||||\u201d custom_padding=\u201d0px|35px|0|0px|false|false\u201d custom_padding_tablet=\u201d|35px|20px|35px||true\u201d custom_padding_phone=\u201d|35px|11px|35px|false|true\u201d animation_direction=\u201dtop\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|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 Le cellule arriveranno congelate su ghiaccio secco ad una concentrazione di circa 5 x 105<\/sup>cellule\/fiala<\/b><\/p>\n [\/et_pb_text][et_pb_text _builder_version=\u201d4.16\u2033 background_size=\u201dinitial\u201d background_position=\u201dtop_left\u201d background_repeat=\u201dripeti\u201d custom_padding=\u201d||0px|||\u201d global_colors_info=\u201d{}\u201d]<\/p>\n 1) Quando le cellule sono stabilizzate e in crescita, iniziare a usare un terreno contenente 0,75 \u03bcg\/ml di puromicina. Continuare a mantenere le cellule in un terreno contenente 0,75 \u03bcg\/ml di puromicina.<\/p>\n 2) Le cellule devono essere divise quando sono confluenti.<\/p>\n 3) Per dividere le cellule (i volumi indicati presuppongono che le cellule siano in un T25):<\/p>\n A. Utilizzando una tecnica sterile, rimuovere il terreno e sciacquare la beuta con 2-3 ml di HBSS sterile. Rimuovere e scartare l'HBSS.<\/p>\n B. Aggiungere 1 ml di tripsina e incubare per 2-3 minuti. Le cellule devono essere controllate al microscopio invertito per determinare se hanno iniziato ad arrotondarsi, sollevarsi e galleggiare. Se le cellule non si sono staccate dopo 3 minuti, \u00e8 possibile incubare altri 1-2 minuti<\/p>\n C. Serrare il tappo e inclinare delicatamente la beuta da un lato all'altro per rimuovere tutte le cellule. La beuta pu\u00f2 essere picchiettata leggermente di lato per allentare le cellule, se necessario.<\/p>\n D. Aggiungere 4 ml di nuovo terreno di coltura alla beuta non appena le cellule galleggiano per inattivare la tripsina. Risciacquare i lati della beuta pi\u00f9 volte con il nuovo terreno di coltura per lavare via tutte le cellule dalla plastica e nella soluzione. Rimuovere tutto il liquido contenente le cellule e trasferire in una beuta conica da 15 ml (o 50 ml se si mettono insieme 3 o pi\u00f9 beute).<\/p>\n E. Utilizzando una tecnica sterile, prelevare un'aliquota per il conteggio su un emocitometro.<\/p>\n F. Mentre si contano le cellule, il resto della soluzione deve essere centrifugato nella centrifuga clinica per 5 minuti a 1000 giri al minuto.<\/p>\n 4) Dopo aver calcolato il conteggio delle cellule, piastrare le cellule a 2,5 x 105<\/sup>\u00a0cellule per pallone con filtro T 25.<\/p>\n 5) Le cellule devono essere alimentate ogni 2-3 giorni con un terreno di coltura fresco contenente 0,75 \u03bcg\/ml di puromicina.<\/p>\n <\/b>Le cellule devono essere congelate a non meno di 5x 105<\/sup>\u00a0<\/span>cellule \/ml\/crioviale\u00a0nei terreni di coltura<\/span>\u00a0<\/span>contenente 10% DMSO e 30% FBS\u00a0<\/span>senza puromicina<\/strong>\u00a0<\/span>e successivamente posto in una camera di congelamento con isopropanolo a -80\u00b0C durante la notte. Trasferire nell'azoto liquido il giorno successivo.<\/p>\n [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d||0px||||\u201d custom_padding_tablet=\u201d|35px||35px||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 [\/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 Professore di ricerca pediatrica Warren Alpert Medical School della Brown University e Dipartimento di pediatria, Hasbro Children's Hospital, Providence, RI Dipartimento di anestesia, Children's Hospital Boston e Harvard Medical School, Boston, MA Direttore medico, The Progeria Research Foundation<\/p>\n Telefono: 978-535-2594 Italiano: [\/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=\u201d4.27.2\u2033 custom_padding=\u201d||0px||\u201d custom_padding_last_edited="off|desktop" hover_enabled="0" global_colors_info="{}" sticky_enabled="0"]<\/p>\n Italiano: [\/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=\u201d4.21.0\u2033 background_color=\u201d#29327a\u201d custom_margin=\u201d-2px|||||\u201d 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{}\u201d][et_pb_row_inner 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|30px||false|false\u201d 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_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d 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 tipo=\u201d1_3\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d global_colors_info=\u201d{}\u201d][\/et_pb_column_inner][et_pb_column_inner tipo=\u201d1_3\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d global_colors_info=\u201d{}\u201d][et_pb_image Italiano: src=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/07\/RFRFinish.png\u201d title_text=\u201dRFRFinish\u201d _builder_version=\u201d4.21.0\u2033 _module_preset=\u201ddefault\u201d custom_margin=\u201d|20px||-50px|false|false\u201d global_colors_info=\u201d{}\u201d][\/et_pb_image][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row column_structure=\u201d1_4,1_4,1_2\u2033 make_equal=\u201don\u201d module_class=\u201d 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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=\u201dbackground-position: center 18% !important;\u201d global_colors_info=\u201d{}\u201d] Protocolli di coltura cellulare immortalata [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 use_custom_gutter=\u201don\u201d gutter_width=\u201d1\u2033 specialty=\u201don\u201d padding_left_1=\u201d35px\u201d padding_left_2=\u201d35px\u201d padding_2_tablet=\u201d|||0px\u201d padding_2_phone=\u201d\u201d padding_2_last_edited=\u201don|desktop\u201d module_class_1=\u201dsidebar-secondary-nav\u201d module_class=\u201dhandprint-bg\u201d _builder_version=\u201d4.16\u2033 background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png\u201d parallax=\u201don\u201d parallax_method=\u201doff\u201d inner_width=\u201d100%\u201d inner_max_width=\u201d100%\u201d custom_padding=\u201d0|0px|54px|0px|false|false\u201d z_index_tablet=\u201d500\u2033 border_width_top=\u201d10px\u201d border_color_top=\u201d#8fd2ed\u201d [\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\nSupporto di crescita<\/h4>\n
Tripsina<\/strong><\/h4>\n
Soluzione salina bilanciata di Hank<\/strong><\/h4>\n
DMSO per il congelamento<\/strong><\/h4>\n
Protocollo per lo scongelamento dei fibroblasti<\/b><\/h4>\n
\n
Subcoltura di linee cellulari PRF<\/b><\/h4>\n
Congelamento:<\/h4>\n
Per ulteriori informazioni contattare:<\/strong><\/h4>\n
Dott. Leslie B. Gordon, dottore in medicina e chirurgia<\/h6>\n
Fax: 508-543-0377
<\/span>lgordon@progeriaresearch.org<\/a><\/p>\nWendy Norris<\/span><\/h6>\n
Telefono: 401-274-1122 x 48063
e-mail: wnorris@brownhealth.org<\/a><\/a><\/span><\/div>\nIscrizione<\/h2>\n
per il nostro<\/h2>\n
Iscriviti alla newsletter!<\/h2>\n
Insieme, noi<\/h2>\n
VOLERE<\/em><\/h2>\n
trova la 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