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本帖最后由 hsution 于 2012-4-10 06:43 编辑 ; g1 A/ k* D$ _
' v) n4 B# s+ a! h" g# a" q7 V缓冻速融是原则
. ?0 ^" V9 {( I% ]) S1). 90% FBS+ 10%DMSO
6 b/ I! Q! e' j4 t, G5 c* C! F2)From Stemgent公司protocol.
- Q0 M# s; O$ L/ T2X miPSC Cryopreservation Medium & |' A) o0 X- w; K f4 n
60 ml DMEM 2 a# B ?5 |( i! p9 Z. e
20 ml FBS (Hyclone) or ES-Qualified, defined FBS # k \! r& m, V- i
20 ml DMSO 8 g. v; [' i' Q+ T5 u2 O* H
Filter-sterilize medium using a 0.22 μm pore size, low protein-binding filter." v: H' C4 U# U. a5 D
Make 2X miPSC Cryopreservation Medium fresh before use and keep on ice at all times.
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: H6 X r' R" i s- yNote: Work quickly once the DMSO-containing 2X miPSC Cryopreservation Medium is added to the cells
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Stemcell 公司CryoStor CS10冻存液 ready to use/ ^. p( e. N" r, e3 l4 Y
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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,
9 {# A7 B& i4 K3 m! }9 h9 t对我个人而言 3)>1)>2)- |# q+ }+ @6 { v: C
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.8 | h- V" D; j1 S% Q
5 U" P9 u* {+ s( D* ] N+ j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):2 |7 F1 U, k) g& Q# m$ K5 Z2 t2 w K
1. Remove the vial of cells from the liquid nitrogen storage tank or -80.
$ W3 f& Q; W. W- r) [) o2. Roll the vial between gloved hands for 3 to 5 seconds to remove the frost. & }0 m- R" h* ^1 G6 j
3. Immerse the vial into a 37°C water bath.
' p1 i/ O: E1 _. ]5 aNote: Do not submerge the cap of the vial in the water bath to prevent possible contamination. 8 e# z3 o3 f' f& F
4. When only a small ice crystal remains, remove the vial from the water bath and spray with ethanol to sterilize. 4 Y8 Q. {+ b0 m# [* S$ U1 M! E2 l& L- A
5. In a sterile biological safety cabinet, transfer the contents of the vial directly to the bottom of a 15 ml conical tube. ) R% j; N( g! P+ }
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. * k/ o+ l) r( P& y( {
7. Centrifuge the cells at 200 x g for 5 minutes at room temperature.$ m5 N" Q/ `/ N- q, }
8. Bring the pelleted cells back to the biological safety cabinet. U0 S) f' I( ?8 Y/ r/ x
9. Carefully aspirate the supernatant from the cell pellet.
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