Cell melting, passing & freezing

Cell melting, passing & freezing

melting written by 김형준

1. dissolve stock(Hela 신 good in LN2). rapidly at 37℃

2. 10㎖ media in a 15㎖ C.tube .

3. dissolve cell mix (1㎖) with 10㎖ media.

4. gently up & down. (to remove DMSO)

5. centrifuge at 1300rpm, 3min, RT.

6. suction of media. (the pellet are cells)

7. add 5㎖ of new media & prepare a 100Ø dish containing 6㎖ media.

8. gently pipetting 17～20 times up&down.

9. add the 5㎖ continnig cells into a 100Ø dish.

10. culture

passing

1. after 24 hour as doing above, have to pass that other dishs.

(when confluency 70～80%)

2. resuspension with 5㎖ media.

․ 5～6 drops into a 100Ø(1ea) . that′s gonna be 40～50% confluency.

․ all of the last into 150Ø(1ea)

3. when the 150Ø is 80% conf. , passing it into 150Ø ,2ea for feezing

(the 100Ø → continue to passing for experiment)

freezing

1. preparing of feezing media.

(10%DMSO , 10% FBS , 1% Antibiotics in DMEM media)

2. cell trypsinization. (at 37℃ , 3min)

․ suction of the old media

․ PBS washing 2ea (5㎖)

․ add 2㎖ Trypsin-EDTA (100Ø dish = 2㎖ 150Ø dish = 5㎖)

․ incubation at 37℃ for 3min.

3. resuspension with feezing media.

․ 100Ø , 80% conf. → feezing media 4㎖

․ 150Ø , 80% conf. → feezing media 8㎖

4. aliquot 1㎖ into cryobial.

5. transfer it into a cryotank which contains isopropyl alcohol.

(decrease 1℃)

6. keep it in the -70℃ deepfreezer. ( 〉4hrs)

7. transfer it into the LN2 tank.