Wash L929 Cells with DPBS (with or w/out CA MG?) - DPBS with or W/out CA,MG? (Feb/22/2010 )

Hi,

I am doing cytotoxicity testing using Bio-Rad DC protein Test kit where L929 mouse fibroblast cells are cultured in 24Well Microplate.

Before the testing, medium are removed and washed with DPBS (Room Temp) 3 times before incubating them with lysate (to lyse the cells)

OK, now, i have been using DPBS (withOUT CA,MG) to wash the cells, and stained the cells to look for the cells distribution layer over the well.
Recently i changed the PBS to PAA brand, and the cells Peels Off (almost half) after washing them.
This problem does not exist previously.

Should i use DPBS with CA,MG? I wont be doing trypsinization at this stage, so CA,MG present won't is not a matter , am i correct?

CA,MG will causes the protein binding more strongly, will this eventually causes the protein content to increase in my protein assay?

Or should i just stick back with my PBS w/out CA Mg, and warmed it before washing?

Please Help!!
THANKS!!

if you want the cells to stick to the plate after washing, use PBS plus Ca and Mg. You should have your control cells to adjust any difference it might cause due to this and use this as reference in your experiment.

Just to check, your 96 well plate is tissue-culture treated right? Also, as mentioned in the link provided by scolix above, use PBS WITH Ca/Mg if you don't want to detach your cells during the wash. (PBS without Ca/Mg or with EDTA is used if you want to lift the cells).