[cell biology work] Primary_cell_culture

[Seung Jae Jeong]

                                                                                                9. 22.20

Primary cell culture

< materials >

1.     A-1X PBS with 2X antibiotics (2ml of Ab/100ml)

2.     Trypsin-EDTA with 2X antibiotics (2ml of Ab/100ml)

3.     10%FBS-EMEM media with 2X antibiotics (10ml of Ab./500ml)

4.     Autoclaved-blue tip & yellow tip

5.     sharp forcepts, scissors, 100% EtOH, Alcohol lamp, Stero-Microscope, illuminator etc…..

 

 

 

1. anethesize the mouse with avertin

2. open the mouse abdomen in clean bench

3. take the uterus into 100mm dish with 20ml of A-1X PBS

4. washing briefly X 2ea

5. separate the each embryo into different 35mm dish with A-1X PBS

6. washing briefly X 2ea

7. remove the placenta and discard

8. uncover the both sacs and colletc into E.tube (for western or PCR)

9. washing briefly X 2ea

10. discard 1X PBS

11. chopping the embryo with mess blade or syringe

12. add 1 – 2ml of Trypsin-EDTA with 2X antibiotics and pipetting (X10ea)

13. transfer the above sol. into 50ml C.tube

    — repeat 5 – 13 steps for each embryo, as soon as possible

14. shaking incubation at 37C, 30min (or 1hr)

15. centrifuge 2000rpm, 5min

16. remove Trypsin-EDTA (dont’t discard pelltet and jelly-like clots)

17. directly add – 6ml of 10%FBS-DMEM containg 2X antibiotics (for small embryo) and pipetting 20ea

                – 12ml of                          –                              and       –

18. transfer the above media into 50mm dish or 100mm dish

19. follow the normal culture method.

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