{"id":1004,"date":"2017-02-27T21:19:33","date_gmt":"2017-02-27T21:19:33","guid":{"rendered":"https:\/\/www.progeriaresearch.org\/?page_id=1004"},"modified":"2025-02-11T09:34:37","modified_gmt":"2025-02-11T14:34:37","slug":"lymphoblast-cell-culture-protocols","status":"publish","type":"page","link":"https:\/\/www.progeriaresearch.org\/de\/lymphoblast-cell-culture-protocols\/","title":{"rendered":"Protokolle f\u00fcr die Lymphoblastenzellkultur"},"content":{"rendered":"<p>[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\/Jes.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=\u201dHintergrundposition: Mitte 18% !important;\u201d global_colors_info=\u201d{}\u201d]<\/p>\n<h1 class=\"vc_custom_heading\" data-fontsize=\"34\" data-lineheight=\"48\" style=\"text-align: right;\">Lymphoblastenzelle<\/h1>\n<h1 class=\"vc_custom_heading\" data-fontsize=\"34\" data-lineheight=\"48\" style=\"text-align: right;\">Kulturprotokolle<\/h1>\n<p>&nbsp;<\/p>\n<p>[\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 use_custom_gutter=\u201dan\u201d gutter_width=\u201d1\u2033 specialty=\u201dan\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=\u201dan|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=\u201dan\u201d parallax_method=\u201daus\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 use_custom_width=\u201dan\u201d width_unit=\u201daus\u201d custom_width_percent=\u201d100%\u201d global_colors_info=\u201d{}\u201d][et_pb_column type=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_sidebar area=\u201det_pb_widget_area_14\u2033 disabled_on=\u201dan|an|aus\u201d module_class=\u201dUnterseiten-Seitenleisten\u201d _builder_version=\u201d4.16\u2033 animation_style=\u201dverblassen\u201d z_index_tablet=\u201d500\u2033 border_width_right=\u201d5px\u201d gesperrt=\u201daus\u201d global_colors_info=\u201d{}\u201d]<br \/>\n[\/et_pb_sidebar][\/et_pb_column][et_pb_column Typ=\u201d3_4\u2033 Spezialspalten=\u201d3\u2033 _builder_version=\u201d4.16\u2033 benutzerdefiniertes_Padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d benutzerdefiniertes_Padding__hover=\u201d|||\u201d][et_pb_row_inner _builder_version=\u201d4.16\u2033 Hintergrundfarbe=\u201d#00b2e2\u2033 benutzerdefinierter_Margin=\u201d41px||\u201d benutzerdefinierter_Margin_Tablett=\u201d0px||\u201d custom_margin_phone=\u201d\u201d custom_margin_last_edited=\u201dam|Telefon\u201d custom_padding=\u201d39.4375px|20px|0|20px|false|true\u201d animation_style=\u201dFolie\u201d animation_direction=\u201doben\u201d animation_intensity_slide=\u201d25%\u201d box_shadow_style=\u201dpreset1\u2033 box_shadow_blur=\u201d38px\u201d box_shadow_spread=\u201d-12px\u201d gesperrt=\u201daus\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=\u201dDie Arbeit der 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|50px||\u201d custom_padding_tablet=\u201d|50px||50px||true\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201dauf|Desktop\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d\u201d header_font_size_last_edited=\u201dauf|Desktop\u201d header_2_font_size_tablet=\u201d25px\u201d header_2_font_size_phone=\u201d\u201d header_2_font_size_last_edited=\u201dauf|Tablet\u201d header_4_font_size_tablet=\u201d\u201d header_4_font_size_phone=\u201d19px\u201d header_4_font_size_last_edited=\u201dauf|Telefon\u201d header_4_line_height_tablet=\u201d\u201d header_4_line_height_phone=\u201d1.2em\u201d header_4_line_height_last_edited=\u201dauf|Telefon\u201d z_index_tablet=\u201d500\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n<h2 style=\"text-align: center;\"><strong>Protokoll zur Kultivierung transformierter Lymphozyten<\/strong><\/h2>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=\u201dam|Telefon\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=\u201doben\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=\u201d4.27.4\u2033 background_size=\u201dinitial\u201d background_position=\u201doben_links\u201d background_repeat=\u201dwiederholen\u201d custom_padding=\u201d||0px||false|false\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033]<\/p>\n<h4 style=\"text-align: center;\"><strong>Protokoll zur Kultivierung von Lymphoblasten<\/strong><\/h4>\n<h4><strong>Wachstumsmedium<\/strong><\/h4>\n<p>RPMI 1640 \u2013 ThermoFisher # 11875<br \/>15% Fetales Rinderserum (FBS) \u2013 ThermoFisher #10437-028<br \/>1% (1X) Penicillin-Streptomycin (optional) \u2013 ThermoFisher #15140-122<\/p>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Allgemeine Hinweise<br \/><\/strong><\/h4>\n<ul>\n<li>Das Wachstumsmedium sollte im Inkubator immer auf 37\u00b0C, 5% CO<sub>2<\/sub> 30 Minuten vor Gebrauch<\/li>\n<li>Benutzen Sie immer Flaschen mit bel\u00fcfteten Verschl\u00fcssen<\/li>\n<li>Bebr\u00fcten Sie den Kolben immer in aufrechter Position<\/li>\n<li>Es sollten nicht mehr als 20 ml in einem T25-Kolben verwendet werden<\/li>\n<\/ul>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Auftauen gefrorener Zellen<br \/><\/strong><\/h4>\n<p>Gefrorene Zellen werden in 1-ml-Aliquots auf Trockeneis geliefert. Bewahren Sie die Zellen gefroren auf, bis sie f\u00fcr die Zellkultur aufgetaut werden, oder legen Sie sie in fl\u00fcssigen Stickstoff, wenn Sie nicht sofort mit der Kultur beginnen. Es wird jedoch empfohlen, dass Sie, wenn Sie die Zellen f\u00fcr eine sp\u00e4tere Verwendung aufbewahren m\u00f6chten, frische Vorr\u00e4te anbauen und mehrere Ampullen einfrieren. Die Zellen werden in 45% RPMI-1640, 50% FBS und 5% DMSO (Gewebekulturqualit\u00e4t) kryokonserviert.<\/p>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Geben Sie 10 ml RPMI-1640, 15% f\u00f6tales Rinderserum (FBS), \u00b1 Antibiotika in eine T-25-Flasche mit bel\u00fcfteter Kappe.<\/li>\n<li>Lassen Sie das Medium in einem 37 \u00b0C befeuchteten Inkubator mit 5% CO ausgleichen<sub>2<\/sub> 30 Minuten an der Luft inkubieren. Die Flaschen sollten aufrecht inkubiert werden.<\/li>\n<li>Tauen Sie jede Ampulle oder jedes Fl\u00e4schchen mit gefrorenen Zellen einzeln in einem 37\u00a0\u00b0C warmen Wasserbad oder einem Becher mit lauwarmem Wasser auf.<\/li>\n<li>Reinigen Sie die Au\u00dfenseite der Ampulle oder des Fl\u00e4schchens schnell mit 70% Ethanol. Wenn sich die Zellen in einer Glasampulle befinden, zerbrechen Sie die Ampulle vorsichtig und sch\u00fctzen Sie dabei Ihre Finger vor Glassplittern.<\/li>\n<li>Entnehmen Sie den 1 ml gefrorener Zellen mit einer sterilen Pipette und geben Sie ihn in eine T-25-Flasche mit 10 ml Medium. Beschriften Sie die Flasche deutlich, um jede Zelllinie zu identifizieren.<\/li>\n<li>Stellen Sie den Kolben in einen 37\u00b0C befeuchteten Inkubator mit 5% CO<sub>2<\/sub> in der Luft. Flaschen sollten in aufrechter Position inkubiert werden.<\/li>\n<li>Nach 24 Stunden 5 ml Medium von oben entfernen, ohne die am Boden des Kolbens abgelagerten Lymphozyten zu st\u00f6ren, und durch 5 ml vorgew\u00e4rmtes Medium ersetzen.<\/li>\n<li>Fahren Sie mit dem Protokoll zur Lymphoblastenzellkultur fort.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Lymphoblastenkultur<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Drei bis vier Tage nach dem Auftauen die Zellen z\u00e4hlen. Die Zellen sind nicht haftend und wachsen in Klumpen. Klumpen durch vorsichtiges Pipettieren aufbrechen.<\/li>\n<li>Zellen z\u00e4hlen.<\/li>\n<li>Idealerweise sollte die Zellkonzentration 2 x 10 nicht \u00fcberschreiten<sup>6<\/sup> Zellen\/ml.<\/li>\n<li>Je nach Zellkonzentration k\u00f6nnen die Zellen erneut zugef\u00fchrt, geteilt oder eingefroren werden.<\/li>\n<li>F\u00fchren Sie die F\u00fctterung durch Zugabe von 5\u20136 ml ausgeglichenem Wachstumsmedium durch.<\/li>\n<li>S\u00e4en Sie neue Kulturen mit mindestens 2 x 10<sup>5<\/sup> Zellen\/ml.<\/li>\n<li>Die Zellen sollten bei etwa 5 x 10 eingefroren werden<sup>6<\/sup> Zellen\/ml. Befolgen Sie das Protokoll zum Einfrieren von Zellen.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Einfrieren von Lymphoblasten<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Die Zellen sollten bei etwa 5 x 10 eingefroren werden<sup>6<\/sup> Zellen\/ml.<\/li>\n<li>\u00dcbertragen Sie das entsprechende Zellvolumen in ein 15- oder 50-ml-konisches R\u00f6hrchen.<\/li>\n<li>10 Minuten lang bei 4\u00a0\u00b0C und 100\u00a0xg zentrifugieren.<\/li>\n<li>Entfernen Sie das Medium und suspendieren Sie es im entsprechenden Volumen kalten Gefriermediums erneut.<\/li>\n<li>\u00dcbertragen Sie jeweils 1 ml Zellen in ein Kryovial.<\/li>\n<li>Legen Sie die Kryor\u00f6hrchen in die Gefrierkammer und stellen Sie die Kammer \u00fcber Nacht in einen -80\u00a0\u00b0C hei\u00dfen Gefrierschrank.<\/li>\n<li>\u00dcberf\u00fchren Sie die Kryor\u00f6hrchen am n\u00e4chsten Tag in fl\u00fcssigen Stickstoff.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner _builder_version=\u201d4.27.4\u2033 _module_preset=\u201ddefault\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033 custom_padding=\u201d24px||24px|||\u201d][et_pb_column_inner saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.27.4\u2033 _module_preset=\u201ddefault\u201d global_colors_info=\u201d{}\u201d][et_pb_button button_url=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2025\/02\/Lymphoblast-protocol-1-22-25.pdf\u201d button_text=\u201dProtokoll zur Kultivierung von Lymphoblasten\u201d admin_label=\u201dProtokoll zur Kultivierung von Lymphoblasten\u201d _builder_version=\u201d4.27.4\u2033 _module_preset=\u201ddefault\u201d button_text_color=\u201d#FFFFFF\u201d button_bg_color=\u201d#00B2E2\u2033 button_border_color=\u201d#00B2E2\u2033 global_colors_info=\u201d{}\u201d][\/et_pb_button][\/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 global_colors_info=\u201d{}\u201d]<\/p>\n<h4><strong>F\u00fcr weitere Informationen kontaktieren Sie bitte:<\/strong><\/h4>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\u201dam|Telefon\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=\u201doben\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=\u201dauf|desktop\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.19.5\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n<h6>Leslie B. Gordon, MD, PhD<\/h6>\n<p>Professor f\u00fcr p\u00e4diatrische Forschung Warren Alpert Medical School der Brown University und Abteilung f\u00fcr P\u00e4diatrie, Hasbro Children&#039;s Hospital, Providence, RI Abteilung f\u00fcr An\u00e4sthesie, Children&#039;s Hospital Boston und Harvard Medical School, Boston, MA Medizinischer Direktor, The Progeria Research Foundation<\/p>\n<p>Telefon: 988-535-2594<br \/>Fax: 508-543-0377<br \/><span> <\/span><a href=\"mailto:lgordon@progeriaresearch.org\" title=\"mailto:lgordon@progeriaresearch.org\">lgordon@progeriaresearch.org<\/a><\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner Typ=\u201d1_2\u2033 gespeicherte_Spezialit\u00e4tsspaltenart=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 benutzerdefiniertes_Padding=\u201d|||20px\u201d benutzerdefiniertes_Padding_Tablet=\u201d20px|||0px\u201d benutzerdefiniertes_Padding_Telefon=\u201d\u201d benutzerdefiniertes_Padding_last_edited=\u201dein|Telefon\u201d 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animation_direction=\u201dleft\u201d animation_intensity_slide=\u201d25%\u201d link_option_url=\u201dhttps:\/\/lp.constantcontactpages.com\/sl\/88gWWwz\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d30px\u201d header_font_size_last_edited=\u201dan|desktop\u201d z_index_tablet=\u201d500\u2033 border_radii=\u201dan|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201dan\u201d button_bg_color__hover=\u201d#8fd2ed\u201d button_border_color__hover_enabled=\u201dan\u201d]<\/p>\n<h2>Melden Sie sich an<\/h2>\n<h2>f\u00fcr unsere<\/h2>\n<h2>Neuigkeiten!<\/h2>\n<p>[\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_cta button_url=\u201dhttps:\/\/progeriaresearch.donorsupport.co\/-\/XZHJVWZR\u201d button_text=\u201dJetzt spenden\u201d admin_label=\u201dGemeinsam werden wir die Heilung finden!\u201d module_class=\u201dsign-btn\u201d _builder_version=\u201d4.16\u2033 header_font_size=\u201d25px\u201d background_color=\u201d#29327a\u201d animation_style=\u201dslide\u201d animation_direction=\u201dleft\u201d animation_intensity_slide=\u201d25%\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d30px\u201d header_font_size_last_edited=\u201don|desktop\u201d body_font_size_tablet=\u201d\u201d body_font_size_phone=\u201d\u201d body_font_size_last_edited=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 border_radii=\u201don|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201don\u201d button_bg_color__hover=\u201d#8fd2ed\u201d button_border_color__hover_enabled=\u201dein\u201d]<\/p>\n<h2>Gemeinsam<\/h2>\n<h2><em>WILLE<\/em><\/h2>\n<h2>Finde die Heilung!<\/h2>\n<p>[\/et_pb_cta][\/et_pb_column][et_pb_column Typ=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\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 Streifen Fu\u00dfzeile Streifen Kopie\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":"<p>[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\/Jes.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=\u201dHintergrundposition: Mitte 18% !important;\u201d global_colors_info=\u201d{}\u201d] Protokolle f\u00fcr Lymphoblasten-Zellkulturen [\/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 Parallaxmethode = \u201eaus\u201c innere Breite = \u201e100%\u201c innere maximale Breite = \u201e100%\u201c benutzerdefinierte Polsterung = \u201e0|0px|54px|0px|false|false\u201c z_index_tablet = \u201e500\u2033 Rahmenbreite oben = \u201e10px\u201c Rahmenfarbe oben = \u201e#8fd2ed\u201c [\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=\"Lymphoblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]<strong>Protocol for Culturing Transformed Lymphocytes<\/strong>\r\n\r\nFrom Shipment of Frozen Cells\r\n\r\nFrozen cells will be received in 1 ml aliquots on dry ice. Keep cells frozen until thawed for cell culture or place in liquid nitrogen storage if not starting cultures right away. However, it is recommended that if you are going to store the cells for later use, grow up fresh stocks and freeze multiple ampoules. The cells are cryopreserved in 45% RPMI-1640, 50% fetal calf serum, and 5% DMSO (tissue culture grade).\r\n<ol>\r\n \t<li>Place 10 ml of RPMI-1640, 15% fetal calf serum (FCS), \u00b1antibiotics in a T-25 flask.<\/li>\r\n \t<li>Allow the media to equilibrate in a 37\u00b0C humidified incubator with 5% CO 2in air for 30 minutes.<\/li>\r\n \t<li>Thaw each ampoule of frozen cells in a 37\u00b0C water bath or a beaker of lukewarm water, one at a time.<\/li>\r\n \t<li>Quickly clean the outside of the ampoule off with a sterile isopropanol prep pad, then wrap the prep pad around the ampoule to protect fingers and crack the seal on the ampoule inside a biological safety hood.<\/li>\r\n \t<li>Remove the 1 ml of frozen cells with a sterile glass Pasteur pipet and place in a T-25 flask with 10 ml of medium. Clearly label flask to identify each cell line.<\/li>\r\n \t<li>Place the cells in a 37\u00b0C humidified incubator with 5% CO 2in the air.<\/li>\r\n \t<li>After 24 hours remove 5 ml of medium from the top without disturbing lymphocytes settled on the bottom of the flask and replace with 5 ml of pre-warmed medium.<\/li>\r\n \t<li>Feed the cells with 5 - 6 ml of fresh medium every 3 - 4 days and split into new cultures as required.<\/li>\r\n<\/ol>\r\n<strong>From Shipment of Live Cultures<\/strong>\r\n<ol>\r\n \t<li>Live lymphocyte cultures will be received in T-25 flasks filled to the top with RPMI-1640, 15% FCS, 1% Penicillin-Streptomycin (Gibco). The caps will be tight and sealed with parafilm.<\/li>\r\n \t<li>Remove parafilm.<\/li>\r\n \t<li>Place cultures in a 37\u00b0C humidified incubator with 5% CO2 in air and allow cells to settle out (20 - 30 minutes).<\/li>\r\n \t<li>Using a sterile pipet remove all but 10 - 15 ml of medium and discard.<\/li>\r\n \t<li>Place caps back on flasks loosened slightly to allow gas exchange (Check to make sure cap threads do not have media on them. If so you may want to transfer to a new flask.) Place the cultures back in the incubator with 5% CO 2in air. They should recover in a day or two.<\/li>\r\n \t<li>Feed the cells with fresh medium every 3 - 4 days by resuspending the cell aggregates and removing 50 -60% of the volume and replacing with fresh media. Cells removed from the flask can be used to initiate new cultures as needed.<\/li>\r\n<\/ol>\r\n<strong>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\n<a href=\"mailto:Leslie_Gordon@brown.edu\">Leslie_Gordon@brown.edu<\/a>\r\n<div><span style=\"font-family: Georgia, Palatino;\">Wendy Norris<\/span><\/div>\r\n<div><span style=\"font-family: Georgia, Palatino;\">PRF Cell and Tissue Bank\r\nPhone: 401-274-1122 x 48063\r\n<a href=\"mailto:wnorris@lifespan.org\" target=\"_blank\" rel=\"noopener noreferrer\">wnorris@lifespan.org<\/a><\/span><\/div>\r\n[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1004","page","type-page","status-publish","hentry"],"yoast_head":"<!-- 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