cos1 cells transfection and 32P label in vivo

[ning xu]

Dear friends,

I have two questions:

1.When I do COS1 cells transiently transfection with pcDNA3RIIB-HA.Befor
transfection the cells density is 60%-70%, 5 hours later the medium was
replaced  with the fresh,complete medium. 72 hours after the start of
the transfection,the cells density is still 60%-70%.I know this results
from the toxitity of the plasmid replication.When you do COS1 cells
transiently transfection with pcDNA3RIIB-HA. How many hours later after
the start of the transfection, you do 32P label in vivo.

2.When you add 2nM Activin A to your labeling cells,Does the serum free
medium with 0.1% BSA?


The following is my protocol for 32P label in vivo,is it OK.

32P label the cell:

Cos1 cells cotransfected with both TGFBRII and TGFBRI
 72hrs after the start of the transfection do  32P label)
 On the 32P label day
 remove medium
Wash cells with DMEM-Phosphate free and serum free medium 3x
Incubate cells with PO4 free and serum free medium for 2-3 hr
 Remove medium
Small flask add 4ml of PO4 free medium+32P*(total of 200uCi )
 In the incubator for 2-3 hr(in perspex box!!1)
Take out small flask and add TGFb(1-10ng/ml) and now medium with 0.1%
BSA
 Leave with cell for 5-15 min
 Remove medium from cells
 Wash cell with ice cold PO4 free medium(medium+50uM Na
orthovanadate—Na3VO4----30ml medium+7.5ul 200mM Na3VO4)
Lysis cells in situ:
   1.1ml ice cold lysis buffer for each flask
   2.stand for 5 min on ice
   3.scrape cells and use the 1ml pipette (tip with filter)
   transfer the cell lysates to ice cold eppendorf tube up down 4x
   4.put cell lysates on ice for 30 min in eppendorfs
   5.spin at 4oC,15,000 25min
   6.collect supernatant=cell lysate extract

Thanking you in advance

Sincerely


ning xu



please reply to my e-mail address:
n.xu at cfi.unsw.edu.au