{"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\/id\/lymphoblast-cell-culture-protocols\/","title":{"rendered":"Protokol Kultur Sel Limfoblas"},"content":{"rendered":"<p>[et_pb_section fb_built=\u201d1\u2033 lebar_penuh=\u201daktif\u201d dinonaktifkan_aktif=\u201dnonaktif|nonaktif|nonaktif\u201d _builder_version=\u201d4.16\u2033 lebar_batas_bawah=\u201d55px\u201d warna_batas_bawah=\u201d#29327a\u201d terkunci=\u201dnonaktif\u201d info_warna_global=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 judul_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=\u201dpada|desktop\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201dpada|ponsel\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dbackground-position: center 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;\">Sel Limfoblas<\/h1>\n<h1 class=\"vc_custom_heading\" data-fontsize=\"34\" data-lineheight=\"48\" style=\"text-align: right;\">Protokol Budaya<\/h1>\n<p>&nbsp;<\/p>\n<p>[\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 gunakan_talang_khusus=\u201daktif\u201d lebar_talang=\u201d1\u2033 spesialisasi=\u201daktif\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=\u201daktif|desktop\u201d kelas_modul_1=\u201dnavigasi-sekunder-bilah-sisi\u201d kelas_modul=\u201dcetakan-bg\u201d versi_pembuat=\u201d4.16\u2033 gambar_latar_belakang=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png\u201d paralaks=\u201daktif\u201d metode_paralaks=\u201dnonaktif\u201d lebar_dalam=\u201d100%\u201d inner_max_width=\u201d100%\u201d bantalan_kustom=\u201d0|0px|54px|0px|false|false\u201d z_index_tablet=\u201d500\u2033 lebar_batas_atas=\u201d10px\u201d warna_batas_atas=\u201d#8fd2ed\u201d gunakan_lebar_kustom=\u201daktif\u201d satuan_lebar=\u201dnonaktif\u201d lebar_kustom_persen=\u201d100%\u201d info_warna_global=\u201d{}\u201d][et_pb_column jenis=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_kustom=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_sidebar area=\u201det_pb_widget_area_14\u2033 dinonaktifkan_aktif=\u201daktif|aktif|nonaktif\u201d kelas_modul=\u201dsubpage-sidebars\u201d _builder_version=\u201d4.16\u2033 gaya_animasi=\u201dmemudar\u201d z_index_tablet=\u201d500\u2033 lebar_batas_kanan=\u201d5px\u201d terkunci=\u201dnonaktif\u201d global_colors_info=\u201d{}\u201d]<br \/>\n[\/et_pb_sidebar][\/et_pb_column][et_pb_column tipe=\u201d3_4\u2033 kolom_khusus=\u201d3\u2033 _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|||\u201d info_warna_global=\u201d{}\u201d bantalan_khusus__hover=\u201d|||\u201d][et_pb_row_inner _builder_version=\u201d4.16\u2033 warna_latar_belakang=\u201d#00b2e2\u2033 margin_khusus=\u201d41px||\u201d tablet_margin_khusus=\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=\u201dKarya Yayasan Penelitian Progeria\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=\u201ddi|desktop\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d\u201d header_font_size_last_edited=\u201ddi|desktop\u201d header_2_font_size_tablet=\u201d25px\u201d header_2_font_size_phone=\u201d\u201d header_2_font_size_last_edited=\u201ddi|tablet\u201d header_4_font_size_tablet=\u201d\u201d header_4_font_size_phone=\u201d19px\u201d header_4_font_size_last_edited=\u201ddi|phone\u201d header_4_line_height_tablet=\u201d\u201d header_4_line_height_phone=\u201d1.2em\u201d header_4_line_height_last_edited=\u201ddi|phone\u201d z_index_tablet=\u201d500\u2033 info_warna_global=\u201d{}\u201d]<\/p>\n<h2 style=\"text-align: center;\"><strong>Protokol untuk Membudidayakan Limfosit yang Telah Ditransformasi<\/strong><\/h2>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner bantalan_khusus_terakhir_diedit=\u201ddi|ponsel\u201d _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|35px|0|0px|false|false\u201d bantalan_khusus_tablet=\u201d|35px||35px||true\u201d bantalan_khusus_ponsel=\u201d\u201d arah_animasi=\u201datas\u201d info_warna_global=\u201d{}\u201d][et_pb_column_inner tipe_kolom_khusus_yang_disimpan=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.4\u2033 ukuran_latar belakang=\u201dawal\u201d posisi_latar belakang=\u201dkiri_atas\u201d pengulangan_latar belakang=\u201dulangi\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>Protokol untuk Budidaya Limfoblas<\/strong><\/h4>\n<h4><strong>Media Pertumbuhan<\/strong><\/h4>\n<p>RPMI 1640 \u2013 ThermoFisher # 11875<br \/>Serum Janin Sapi (FBS) 15% \u2013 ThermoFisher #10437-028<br \/>1% (1X) Penisilin-Streptomisin (opsional) \u2013 ThermoFisher #15140-122<\/p>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Catatan Umum<br \/><\/strong><\/h4>\n<ul>\n<li>Media pertumbuhan harus selalu diseimbangkan dalam inkubator pada suhu 37\u00b0C, 5% CO<sub>2<\/sub> selama 30 menit sebelum digunakan<\/li>\n<li>Selalu gunakan botol dengan tutup berventilasi<\/li>\n<li>Selalu inkubasi labu dalam posisi tegak<\/li>\n<li>Tidak lebih dari 20 ml harus digunakan dalam labu T25<\/li>\n<\/ul>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Mencairkan Sel Beku<br \/><\/strong><\/h4>\n<p>Sel beku akan diterima dalam alikuot 1 ml pada es kering. Simpan sel beku hingga dicairkan untuk kultur sel atau simpan dalam penyimpanan nitrogen cair jika tidak segera memulai kultur. Namun, jika Anda akan menyimpan sel untuk penggunaan selanjutnya, sebaiknya buat stok segar dan bekukan beberapa ampul. Sel dikriopreservasi dalam 45% RPMI-1640, 50% FBS, dan 5% DMSO (tingkat kultur jaringan).<\/p>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Tempatkan 10 ml RPMI-1640, 15% fetal bovine serum (FBS), \u00b1 antibiotik dalam labu T-25 dengan tutup berventilasi.<\/li>\n<li>Biarkan media mencapai keseimbangan dalam inkubator humidifikasi 37\u00b0C dengan 5% CO<sub>2<\/sub> di udara selama 30 menit. Labu harus diinkubasi dalam posisi tegak.<\/li>\n<li>Cairkan setiap ampul atau botol kecil berisi sel beku dalam air bersuhu 37\u00b0C atau gelas kimia berisi air hangat, satu per satu.<\/li>\n<li>Bersihkan bagian luar ampul atau vial dengan cepat menggunakan etanol 70%. Jika sel berada dalam ampul kaca, pecahkan ampul dengan hati-hati dan pastikan jari terlindungi dari pecahan kaca.<\/li>\n<li>Ambil 1 ml sel beku dengan pipet steril dan tempatkan dalam labu T-25 dengan 10 ml medium. Beri label pada labu dengan jelas untuk mengidentifikasi setiap lini sel.<\/li>\n<li>Tempatkan labu dalam inkubator humidifikasi 37\u00b0C dengan 5% CO<sub>2<\/sub> di udara. Labu harus diinkubasi dalam posisi tegak.<\/li>\n<li>Setelah 24 jam, keluarkan 5 ml media dari atas tanpa mengganggu limfosit yang mengendap di dasar labu dan ganti dengan 5 ml media yang telah dihangatkan sebelumnya.<\/li>\n<li>Lanjutkan dengan protokol kultur sel limfoblas.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Kultur Limfoblas<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Tiga hingga empat hari setelah pencairan, hitung sel-selnya. Sel-sel tidak melekat dan tumbuh dalam gumpalan. Pecahkan gumpalan dengan pipet yang lembut.<\/li>\n<li>Hitung sel.<\/li>\n<li>Idealnya, konsentrasi sel tidak boleh melebihi 2 x 10<sup>6<\/sup> sel\/ml.<\/li>\n<li>Bergantung pada konsentrasi sel, isi ulang, pisahkan, atau bekukan sel.<\/li>\n<li>Beri makan kembali dengan menambahkan 5-6 ml media pertumbuhan yang telah diseimbangkan.<\/li>\n<li>Tanam benih kultur baru minimal 2 x 10<sup>5<\/sup> sel\/ml.<\/li>\n<li>Sel harus dibekukan pada suhu sekitar 5 x 10<sup>6<\/sup> sel\/ml. Ikuti protokol pembekuan sel.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>Pembekuan Limfoblas<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>Sel harus dibekukan pada suhu sekitar 5 x 10<sup>6<\/sup> sel\/ml.<\/li>\n<li>Pindahkan volume sel yang sesuai ke dalam tabung kerucut 15 atau 50 ml.<\/li>\n<li>Sentrifus pada suhu 4\u00b0C pada 100 xg selama 10 menit.<\/li>\n<li>Keluarkan medium dan suspensikan kembali dalam volume medium pembeku dingin yang sesuai.<\/li>\n<li>Pindahkan masing-masing 1 ml sel ke dalam kriovial.<\/li>\n<li>Letakkan kriovial dalam ruang pembekuan dan letakkan ruang tersebut dalam freezer bersuhu -80\u00b0C semalaman.<\/li>\n<li>Pindahkan kriovial ke nitrogen cair keesokan harinya.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<p>Bahasa Indonesia: [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner _versi_pembuat=\u201d4.27.4\u2033 _preset_modul=\u201ddefault\u201d hover_enabled=\u201d0\u2033 info_warna_global=\u201d{}\u201d sticky_enabled=\u201d0\u2033 bantalan_khusus=\u201d24px||24px|||\u201d][et_pb_column_inner tipe_kolom_khusus_yang_disimpan=\u201d3_4\u2033 _versi_pembuat=\u201d4.27.4\u2033 _preset_modul=\u201ddefault\u201d info_warna_global=\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=\u201dProtokol untuk Membudidayakan Limfoblas\u201d admin_label=\u201dProtokol untuk Membudidayakan Limfoblas\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 Bahasa Indonesia: _builder_version=\u201d4.16\u2033 bantalan_kustom=\u201d||0px|||\u201d bantalan_kustom_tablet=\u201d|35px||35px||true\u201d bantalan_kustom_ponsel=\u201d\u201d lebar_batas_atas=\u201d10px\u201d warna_batas_atas=\u201d#8fd2ed\u201d info_warna_global=\u201d{}\u201d][et_pb_column_inner tipe_kolom_khusus_simpanan=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_kustom=\u201d|||\u201d info_warna_global=\u201d{}\u201d bantalan_kustom__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.16\u2033 info_warna_global=\u201d{}\u201d]<\/p>\n<h4><strong>Untuk Informasi Lebih Lanjut Silakan Hubungi:<\/strong><\/h4>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner struktur_kolom=\u201d1_2,1_2\u2033 bantalan_khusus_terakhir_diedit=\u201ddi|ponsel\u201d _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|35px|0|0px|false|false\u201d bantalan_khusus_tablet=\u201d|35px||35px||true\u201d bantalan_khusus_ponsel=\u201d\u201d arah_animasi=\u201datas\u201d info_warna_global=\u201d{}\u201d][et_pb_column_inner jenis=\u201d1_2\u2033 jenis_kolom_khusus_tersimpan=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|20px||\u201d custom_padding_tablet=\u201d||20px\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201ddi|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, Dokter Spesialis Bedah Mulut dan Maksimal<\/h6>\n<p>Profesor Riset Pediatri Warren Alpert, Fakultas Kedokteran Universitas Brown dan Departemen Pediatri, Rumah Sakit Anak Hasbro, Providence, RI, Departemen Anestesi, Rumah Sakit Anak Boston dan Fakultas Kedokteran Harvard, Boston, MA, Direktur Medis, Yayasan Riset Progeria<\/p>\n<p>Telepon: 978-535-2594<br \/>Telp.: 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 type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_kustom=\u201d|||20px\u201d bantalan_kustom_tablet=\u201d20px|||0px\u201d bantalan_kustom_phone=\u201d\u201d bantalan_kustom_last_edited=\u201daktif|ponsel\u201d info_warna_global=\u201d{}\u201d bantalan_kustom__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 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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=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 border_radii=\u201daktif|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201daktif\u201d button_bg_color__hover=\u201d#8fd2ed\u201d button_border_color__hover_enabled=\u201daktif\u201d]<\/p>\n<h2>Mendaftar<\/h2>\n<h2>untuk Kami<\/h2>\n<h2>Pembaruan!<\/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=\u201dDonasi Sekarang\u201d admin_label=\u201dBersama-sama, kita akan menemukan obatnya!\u201d modul_kelas=\u201dtanda-btn\u201d _builder_version=\u201d4.16\u2033 ukuran_font_header=\u201d25px\u201d warna_latar belakang=\u201d#29327a\u201d gaya_animasi=\u201dslide\u201d arah_animasi=\u201dkiri\u201d intensitas_animasi_slide=\u201d25%\u201d ukuran_font_header_tablet=\u201d\u201d ukuran_font_header_phone=\u201d30px\u201d ukuran_font_header_last_edited=\u201dpada|desktop\u201d ukuran_font_body_tablet=\u201d\u201d ukuran_font_body_phone=\u201d\u201d ukuran_font_body_last_edited=\u201dpada|desktop\u201d z_index_tablet=\u201d500\u2033 radius_batas=\u201dpada|25px|25px|25px|25px\u201d info_warna_global=\u201d{}\u201d warna_latar_belakang_tombol__hover_enabled=\u201dpada\u201d tombol_warna_latar_belakang_hover=\u201d#8fd2ed\u201d tombol_warna_batas_belakang_hover_diaktifkan=\u201daktif\u201d]<\/p>\n<h2>Bersama-sama, kita<\/h2>\n<h2><em>AKAN<\/em><\/h2>\n<h2>temukan obatnya!<\/h2>\n<p>[\/et_pb_cta][\/et_pb_column][et_pb_column tipe=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|||\u201d Bahasa Indonesia: 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 strip footer strip copy\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 lebar_penuh=\u201daktif\u201d dinonaktifkan_aktif=\u201dnonaktif|nonaktif|nonaktif\u201d _builder_version=\u201d4.16\u2033 lebar_batas_bawah=\u201d55px\u201d warna_batas_bawah=\u201d#29327a\u201d terkunci=\u201dnonaktif\u201d info_warna_global=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 judul_font=\u201d|||||||||\u201d judul_font_size=\u201d55\u2033 warna_latar_belakang=\u201d#29327a\u201d gambar_latar_belakang=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg\u201d posisi_latar_belakang=\u201dtengah_kiri\u201d bantalan_khusus=\u201d9vw||9vw||benar\u201d bantalan_khusus_tablet=\u201d\u201d bantalan_khusus_ponsel=\u201d|56px||\u201d bantalan_khusus_terakhir_diedit=\u201dpada|desktop\u201d ukuran_font_judul_tablet=\u201d45px\u201d ukuran_font_judul_ponsel=\u201d40px\u201d ukuran_font_judul_terakhir_diedit=\u201dpada|ponsel\u201d indeks_z_tablet=\u201d500\u2033 css_utama_element=\u201dposisi_latar_belakang: tengah 18% !penting;\u201d global_colors_info=\u201d{}\u201d] Protokol Kultur Sel Limfoblas [\/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 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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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