Plan for podocytes cell line:
· 1 vial contains 1.5ml, to a total of 2.25 x 106 cells
· Passage number upon arrival is 23. Each batch can be used until passage 40
Thawing from cryovial upon arrival
1) Incubate RPMI media in hood. Have tissue with ethanol ready.
2) Transfer 8ml media into 15ml falcon tube. Transfer 10ml media into T25 flask.
3) Thaw cryovial in waterbath until small clump ice remains.
4) Transfer all contents of cryovial into 15ml falcon tube-à resuspend gently up and down.
5) Spin down at 1500rpm for 3 minutes à discard supernatant.
6) Take 5ml out of T25 and use this to resuspend the pellet in falcon tube.
7) Transfer cell suspension into T25 (have remaining 5ml) à slowly resuspend.
8) Transfer 5ml suspension into another T25 flask.
9) Incubate at 33 degrees.
Once 80% confluency is reached in T25 (average yield is 2 x 106 cells),
1) Incubate RPMI in hood, trypsin in hood, 50ml FBS in hood.
2) Remove media using suction.
3) Wash with 2.5ml 1X PBS.
4) Remove PBS with suction.
5) Add 1.5ml trypsin, tap gently for 3 minutes.
6) Check under microscope for detachment.
7) Neutralise with 3ml media and flush detach cells.
8) Transfer contents into 15ml falcon tube.
9) Centrifuge 1500 rpm for 3 minutes. Remove supernatant and resuspend pellet in 5ml media.
10) Take small aliquot to count cells/ml.
11) Since 1 x 104 cells/cm2 density is recommended for whichever plate/flask used,
12) For T25, use 1 flask: 1 flask for maintenance
13) 25cm2 worth of cells needed: 25 x 104 cells needed
14) (x cells/ml) / (25 x 104 cells): volume needed to seed one t25 flask. Aliquot calculated cell suspension volume into T25.
15) Add 5ml media into T25.
16) Store at 33 degrees.
17) For T75, use 1 flask: for subsequent freezing down.
18) 75cm2 worth of cells needed: 75 x 104 cells needed
19) (x cells/ml) / (75 x 104 cells): volume needed to seed one t75 flask. Aliquot calculated cell suspension volume into T75.
20) Add 20ml RPMI media into T75.
21) Store at 33 degrees.
Once 80 % confluency is reached in T75 (average yield is 7 x 106 cells),
22) Incubate RPMI in hood, trypsin in hood, 50ml FBS in hood.
23) Remove media using suction.
24) Wash with 10ml 1X PBS.
25) Remove PBS with suction.
26) Add 3.0ml trypsin, tap gently for 3 minutes.
27) Check under microscope for detachment.
28) Neutralise with 10ml media and flush detach cells.
29) Transfer contents into 50ml falcon tube.
30) Centrifuge 1500 rpm for 3 minutes. Remove supernatant and resuspend pellet in 10ml media.
31) Take small aliquot to count cells/ml.
32) Since 1 x 104 cells/cm2 density is recommended for whichever plate/flask used,
33) For 6cm flat dish, use 2 dishes: 1 for Western Blot of proliferating cells, 1 for Western Blot of differentiated (eventually) cells
34) 20cm2 x 2 = 40cm2 worth of cells needed
35) 40 x 104 cells needed
36) (x cells/ml) / (40 X 104 cells): volume needed to seed two 6cm flat dish. Divide volume by 2 and aliquot into each flat dish.
37) Add 5ml media into each flat dish.
38) Mark dish meant to differentiate and proliferate.
39) Incubate at 33.
40) Calculate total cells left in 50ml cell suspension (step 30)
· X cells/ml x (10ml – aliquot taken at step 36) = total cells present
· Total cells present in cell suspension / (5 x 105 cells per vial) = vials possible
41) Prepare amount of freeze medium:
· 2.5ml RPMI media x no of vials =
· 2.5ml FBS x no of vials =
· 250ul DMSO x no of vials =
42) Centrifuge at 1500 rpm for 3 minutes of remaining suspension in falcon tube.
43) Remove supernatant.
44) Add required amount of freeze medium to resuspend pellet.
45) Aliquot 1ml of resuspended cells into each cryovial.
46) Label vial passage number, date, cell line.
47) Place vial in box: store overnight at -80 degrees.
48) Transfer to liquid nitrogen next day.
Hello
You have a good protocol, Also you can see the discussion in the same issues and look at the Protocol in attachment
Good Luck
https://www.researchgate.net/post/Protocol_for_maintaining_podocyte_cell_line-opinions
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