Washing buffer - cold or warm? - (Aug/22/2006 )
This is another simple question from someone pretty new to the field. I notice some protocols indicate that they wash cells with cold buffers (PBS etc). Is there any particular reason for this? Should I be washing my cells with cold buffers or warm buffers? Won't washing cells with cold buffers increase the environmental 'shock' applied to cells?
Thanks in advance for answering this.
We wash cells with cold PBS before extracting protein. But we usually, levae the plate with cells on ice bucket for a few min. to cool the cells down to reduce cell shock and to reduce activity of proteases and other enzymes.
But we were to wash cells before trypsinising, we use warm PBS.
well if u are going to wash ur cells for the purpose of passing them use all of ur stuff warmed ( i mean also media trypsin.......) to room
temperature .
But if u are washing ur cells before lysis for protein extraction use cold wash to decrease the effect of protelysis.
hope this will help.
Thanks for the replies! That helped clear some doubt. =)
When passaging cells - my Trypsin/PBS is usually cold, though I put the cells in the incubator for 3-5 mins after adding it. The media I use following this has been warmed to 37oC. I've never had any problems with my cells. (293T, MSC, DPSC, HSC)
Our lab stores all the PBS (minus trypsin) at 4 degrees. Would it be ok to store it at room temperature so I wouldn't have to wait for it to warm up?
We keep all of our PBS at room temperature. I've never had a problem with it.
Now that I think about it..... I'm not sure why we put it in the fridge. Maybe because there aren't any reagent storage shelves in our culture room - only refridgerators.
