ES and TS cell freezing/thawing胚胎干细胞冻存复苏

肖爱华

ES and TS cell freezing/thawing Nagy Lab,Samuel Lunenfeld Research Institute, Room 881 ,Mount Sinai Hospital
  n* a  \2 o5 w/ r4 @该实验室专著于小鼠遗传学以及其跟人类疾病关系 胚胎干细胞冻存复苏 ! R' U3 d! n4 A. k0 _

% J3 d- b# g8 {; zES and TS cell freezing/thawing% I2 L: r2 j' T$ X. c9 o: |( h

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# E4 F* L& z5 zNeeded:& f% E. H+ u% v# L/ N0 @+ O# A
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ES cell freezing medium (2x)0 D$ I+ S$ I; A9 m9 Z3 ?( L$ Y4 m2 I
2x ES cell freezing medium should be made up fresh each time it is to be used, and should comprise freshly prepared 60% DMEM+, 20% FCS, and 20% DMSO (Sigma, Cat. No.D-5879).
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, u5 _0 T# q# {5 x5 a# O5 J) p4 CFreezing in cryovials
( v. Z3 a# G, P# K  nThe general protocol for freezing cells grown in a standard 10 cm dish at 70% confluency is given below:
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1. Change media 2-3 hours before freezing the cells. 0 E( B9 f5 Y5 ?, `

9 [% `7 f* {9 E+ Y2 a/ J3 T2. Freshly prepare 2x freezing media.
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5 `1 t0 P" C% M0 [4 y3. Harvest the cells in a 15 ml. tube containing DMEM+ medium after trypsinization. - b& Z6 c+ X0 ]' b, ^! Q& ^
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4. Spin down at 1000 rpm for 5 min at room temperature. ! H8 a* B) L/ `

5 R0 `" L2 A6 d. S& Q. s5. Remove the supernatant then one or two drops (200 microliters) of DMEM+ medium to the tube. Shake gently but thoroughly, to disperse the cells. ) i/ ]3 E+ ?% {+ Q% `" e

0 q! [2 M6 q0 q% z& }& V6. Add an additional DMEM+ medium to a total volume of 1.5 ml and disperse the cells carefully so that they comprise a single cell suspension.
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7. Add an equal volume (1.5 ml) of 2x freezing medium and mix by pipetting several times.
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  J7 D9 i  m: N! D/ w1 \9 b8. Quickly aliquot the cell suspension into three vials and immediately place them in a Styrofoam box (this will allow them to cool down gradually). Alternatively
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special boxes dedicated to this task can be purchased from a number of manufacturers (for example Stratagene).
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/ z! V' x- \: ~$ w, @, s4 z. l9. Place the box in a -70 0C freezer for 1-2 days, then transfer the individual cryovials into a liquid nitrogen container for long term storage. ( P- W' K5 q( P3 d( {/ ?
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+ X  v4 D: v- z7 l5 ~4 mFreezing in 96 well plates
) o. A  r+ @! h* H! o1. Working one row at a time using a multichannel pipettor change the medium 2-3 hours prior to freezing.
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. ^. O4 }' @3 ]# v! O" _! l2. Freshly prepare 2x cell freezing media.
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- c  W2 r& A# F3. Aspirate the medium from each well and wash the cells with PBS (approximately 200 ml). 8 h7 o8 L9 M, H4 x0 g

9 K2 N, m; ^! \2 H+ l( i# ~4. Add 50 microliter trypsin to each well, then place plate in an incubator for 5-10 min. , L5 w: [  l- H

' Y) }4 G! _8 k+ Z5. Working on ice, preferably in a wide flat container, aliquot 50 microliter of DMEM+ medium into each well. Pipette the cells several times so as to get them ! H2 I- K8 \- a. a2 ]# z) j$ h

! B- a. x) V% v' l6 U8 cinto a homogenous suspension. % X) @  o8 j0 z2 C

6 j( B8 C- @7 M+ `4 {: h1 l6. Then add 100 microliter 2x cell freezing media to the wells, and again pipette to mix. ( j7 n. Y3 |% g+ q6 r* Q( b

. d& D! a! S8 b1 l( [  m7. Finally add 80-100 microliter sterile mineral oil (Sigma, Cat. No. M-8410) to cover the cell/freezing medium mixture.
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8. Wrap the plates in parafilm, place in a styrofoam box, and store in a -70 0C freezer until suchtime as the desired clones have been identified and need to be
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recovered.

新人王

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pingzhou1981

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生物化学zy

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he_liang

谢谢！非常实用！

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