Do RAW cells (high passage number) lyse in PBS? - (May/22/2008 )
I'm doing a Salmonella invasion 24-hr timepoint experiment on RAW 264.7 macrophage cells (passage 35)
I"m expecting number of viable Salmonella that replicate within the macrophages to increase at about 6-10 hrs after invasion. I have gotten results consistent with that before, but recently, at 6 hrs, the numbers of Salmonella have plummetted instead.
To extract the Salmonella within the cells, I wash the cells 3x with PBS, then lyse it using 1% Triton. I suspect that the PBS is lysing the cells because the number of bacteria in the third wash is greater than the number in the initial supernatant.
Anybody have any suggestions as to what is happening or has experienced this before?
Thanks.
hi,
where does the PBS come from? Supplier, home-made?
we had this problem with PBS+Tween 20; an error of our technician...
Seb
I"m expecting number of viable Salmonella that replicate within the macrophages to increase at about 6-10 hrs after invasion. I have gotten results consistent with that before, but recently, at 6 hrs, the numbers of Salmonella have plummetted instead.
To extract the Salmonella within the cells, I wash the cells 3x with PBS, then lyse it using 1% Triton. I suspect that the PBS is lysing the cells because the number of bacteria in the third wash is greater than the number in the initial supernatant.
Anybody have any suggestions as to what is happening or has experienced this before?
Thanks.
We use J774 murine macrophages in our salmonella invasion assay. They are identical to RAW in nearly every way. PBS WILL NOT LYSE YOUR CELLS.
Check that the culture media does NOT contain Pen/strep
Make sure that your OD is correct....maybe your initial MOI is out.
The RAWS should be stable over 100's of passages.....they are for us.
I really cannot think of anything else.
Kindest regards
Rhombus