In cell culture test experiment on the experimental apparatus, culture medium and so on work will cost a lot of manpower and physical strength.
Fortunately, had long been save the experimental steps, who invented the cells to the preservation of a temporary extra cells, to keep the cell vitality to the greatest extent.
1 a, cell cryopreservation.
Material:
the cultured cells grew well, fresh medium, DMSO (
Sigma D-
2650).
, sterile plastic tube cryopreservation (
耐尔根5000 -
0020).
, 0.
4% (
w/v)
台盼蓝(
GibcoBRL15250-
061).
, blood count plate and cover glass, cells cryopreserved instrument (
Also known as program temperature drop, the brand)
2.
Cryopreservation method:
(
1)
Traditional methods: cold storage tube in 4 ℃ - 10 minutes
- - - - - -
- - - - - -
>
- - - - - -
- 20 ℃ for 30 minutes
- - - - - -
- - - - - -
>
-
16 ~ 18 hours (80 ℃
Or overnight)
-
-
-
>
Liquid nitrogen tank vaporphase long-term storage.
-
20 ℃ shall not exceed 1 hour, to prevent excessive intracellular ice crystal, causing a large number of cell death, directly into the - also can skip this step
Of the 80 ℃ refrigerator, but slightly lower.
(
2)
Cool program: program USES has been set to uniform cells cryopreserved instrument to -
1 ~ -
3 ℃ / min speed by room temperature down to (
-
Below 80 ℃)
-
120 ℃, and then put in liquid nitrogen tank vaporphase long-term storage.
Applicable to suspension type and preservation of hybridoma cells.
3.
Steps:
(
1)
24 - before freezing
48 hours to replace half or full amount culture medium, the cell in exponential phase.
(
2)
Cryopreservation solution (
Preparation before use)
: the other in a centrifuge tube, chase to join the culture medium, serum, drop join dimethyl sulfoxide (
DMSO)
Concentration of 20%, that is, make double freeze-stored liquid, at room temperature.
(
3)
Centrifugal collection, culture of cells in serum is medium weight suspended cells, cell suspension (a little amount
About 0.
1毫升)
Survival rates before counting cell concentration and frozen.
(
4)
Take the same amount of freeze-stored liquid and cell suspension, chase a slow drop join cell suspension, and shaking in vitro, made suspension cells cryopreserved (
DMSO final concentration of 5-10%)
, make the cell concentration of 1 ~ 5 & times;
106 cells/ml, mixing, packing in marked the cryopreservation of tube completely, 1 ~ 2 ml/vial, and take a small amount of cell suspension for pollution detection.
After sealed tight, indicating the name of cells, algebra, date.
Then for frozen storage.
