冻存方法

1．细胞：选对数生长期细胞，收集细胞24小时前换液一次。

2．计数：按常规方法把细胞制成细胞悬液，计数，令细胞密度达5×106/ml，离心去上清，吸入离心管中。

3．冻存液：先用培养液9分＋DMSO 1分（或甘油）配成10％的DMSO冻存液，按上法一滴滴加入离心管中，然后用吸管轻轻吹打令细胞重悬。

4．分装：分装入无菌安剖中，每安剖加1.5毫升细胞悬液。

5．封口：用塑料安剖时拧紧瓶口即可；如用火焰封闭安剖口后，仔细检查，定要封严，必要时可浸入蓝色液中观察，为安全起见，把安剖缝入纱布小袋中，以防液氮浸入后，融解时因受热发生爆炸伤人；纱袋一端系以线绳，末端扎有小牌，注明：细胞名称，冻存日期，以便日后查找。

复苏方法

1．从液氮罐中取出安剖；有时因安剖未封严，浸入了液氮，取出后因安剖内液氮迅速气化而发生爆炸，因此应带有防护眼睛和手套。

2．迅速放入盛有36℃～37℃水的搪瓷罐中，扣上盖并不时摇动，尽快解冻。

3．剪开纱布口袋，取出安剖，用70％酒精擦拭消毒后，净化台上打开盖，用吸管吸出细胞悬液，装入离心管中，再补加10ml培养液，吹打使细胞悬浮。

4．低速离心（500～1000转/分）5分钟，取上清后再重复用培养液漂洗、离心。

5．加入培养液适当稀释后，再移装入培养瓶中，置温箱培养，次日更换一次培养液后再继续培养。

Freezing Cells

Contributor: Suprya Jayadev


1) Keep prepared solutions on ice.

2) Determine total cell count of cells to be frozen. (e.g. 1 X 108 )

3) Determine number of vials to be frozen. (e.g. 10 vials at 1 X 107 )

4) Centrifuge cell suspension.

5) Resuspend cell pellet in half the required volume of freezing media. (eg if freezing 10 vials, add 5 mls freezing media)

6) Keep cell suspension on ice.

7) Slowly add dropwise with mixing an equal volume of DMSO solution.

8) Dispense 1 ml of final mixture per sterile Nunc freezing vial.

9) Freeze in liquid nitrogen plug or in controlled rate Planar freezer.

Freezing media

60 ml RPMI1640 or DMEM
40 ml Gammaglobulin free Horse Serum (or FCS)
1 ml Penicillin - Streptomycin

- Filter through 0.2µ filter.
- Aliquot in 15 ml conical tubes and store in -200 C freezer for long term periods.
- Aliquots can be stored in tissue culture flasks in refrigerator for short periods.


20% DMSO solution MAKE FRESH ON DAY OF USE!

If using 100% DMSO:
4 ml pH adjusted medium RPMI (orange-red color)
1 ml sterile 100% DMSO
0.1 ml versene

- Filter through a 0.2 µ filter.

If using 50% DMSO:
3 ml pH adjusted medium
2 ml 50% DMSO
0.1 ml versene

- Filter through a 0.2 µ filter.

NOTE: Versene can be left out, but adding it prevents cells from clumping.