本帖最后由 hsution 于 2012-4-10 06:43 编辑 0 @1 l2 t) H5 [; P8 E! m% n# v! _
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缓冻速融是原则 # E9 D( k* n& U$ n/ b0 S1). 90% FBS+ 10%DMSO, n5 j8 ^6 b. N5 @1 G+ x$ K+ F
2）From Stemgent公司protocol.6 }- s# U! I7 `* F8 a# y6 @
2X miPSC Cryopreservation Medium d [, o J( H
60 ml DMEM , [; w1 i3 c4 t w6 m/ ]20 ml FBS (Hyclone) or ES-Qualified, defined FBS 0 j! c" ^6 X5 z. B20 ml DMSO ~1 y* M. u1 [5 S( K( EFilter-sterilize medium using a 0.22 μm pore size, low protein-binding filter.3 Q7 i" L5 A8 Y' D/ o) D
Make 2X miPSC Cryopreservation Medium fresh before use and keep on ice at all times. * F, [1 C x3 |( Q: g
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Note: Work quickly once the DMSO-containing 2X miPSC Cryopreservation Medium is added to the cells * j# z {2 p! l4 F9 {* m1 t7 ?* I! K2 g* y v
3）. 6 k: s. m, o) j a8 e. UStemcell 公司CryoStor CS10冻存液 ready to use4 L/ X& b/ l0 t2 j
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以上三种冻存液我都用过，感觉差别不大，一定要排个one two three,5 p% K8 Q7 F+ f
对我个人而言 3）>1）>2） ' T7 x/ J6 y3 j y4 t) T! t根据经济情况自行考虑选谁 哈，毕竟现成的公司货方便好用 但它贵. : `; H7 s, e6 y4 _# P- ^: m3 ~- M
复苏我是按stemgent的miPSC复苏protocol操作, 另外个人习惯取冻存的细胞之前，准备好水浴 超净台里的一切 包括管子（Ready to use的状态 节省时间），自带冰盒（有时干冰有时用普通冰），如果去取细胞的路途较长，则自带一烧杯37-8度的水，边走边融，<5min, 2-3min最好，融到管内还有一点点小冰坨即可后续操作（速融）： 8 T, n2 z0 ], F0 K& o5 J1. Remove the vial of cells from the liquid nitrogen storage tank or -80. 1 @# w6 H, W9 g9 K$ ]- U2. Roll the vial between gloved hands for 3 to 5 seconds to remove the frost. , i' d8 N0 u7 W [: Q
3. Immerse the vial into a 37°C water bath. 0 b7 s/ Q* E3 h4 _0 f) x2 V+ V6 j% ZNote: Do not submerge the cap of the vial in the water bath to prevent possible contamination. 8 ^: s, ]5 t' i, F3 W
4. When only a small ice crystal remains, remove the vial from the water bath and spray with ethanol to sterilize. # Z/ V3 ^/ T5 ^4 g* e7 C/ b5. In a sterile biological safety cabinet, transfer the contents of the vial directly to the bottom of a 15 ml conical tube. , |, A) _, i3 F2 I4 U: f
6. Add 5 ml of pre-warmed Culture Medium slowly while gently mixing the contents of the tube. Adding medium slowly will reduce osmotic shock to the cells. $ j9 |& Q" l; Q. D) I8 {- l- D8 A
7. Centrifuge the cells at 200 x g for 5 minutes at room temperature. ) l/ w8 F' @9 D4 N8. Bring the pelleted cells back to the biological safety cabinet. " b5 e. w+ f! Q9 j9. Carefully aspirate the supernatant from the cell pellet. , p2 R+ h" P: i+ {.... . N1 X( q) n+ f/ j5 V希望能有所帮助，尝试一下 必有一款适合您作者: 漂泊的风 时间: 2012-4-10 08:31
你血清的浓度太低了，我们一般用的为50%的浓度，你的太低了，会使细胞膜破裂导致细胞死亡 n: q3 u) k" d t+ b& N" W 作者: lxm5668 时间: 2012-4-10 08:55
谢谢 是不是用90%的FBS冻存，复苏的时候不好融化呢
第一点：冻存液要和原来细胞培养液基本一致 P! b+ G8 S. T2 I