According to the washing solution buffer that is used in cell staining of the flow cytometer. it should be (PBS+5% heat-inactivated FBS +0.1%NaN3).
Can I use PBS + 5% non-heat inactivated serum without 0.1%NaN3 as washing solution buffer instead?
Thank you
I routinely use PBS with either 1% BSA or 1% HSA without azide for washing cells for FACS; however, I would not use FBS that is not heat inactivated for washing stained cells for a very specific reason - untreated serum contains the serum complement system which is activated by antigen-antibody complexes [which by definition stained cells have all over their surfaces] to lyse the cells with the antibody complexes. Heat treatment [56oC for 30 minutes] inactivates one of the serum complement proteins [I think it's C4] and thereby avoids that problem. If you use untreated serum, it can lyse your cells of interest.
I had this happen when I was trying to stain cells for the H-Y antigen for assessing male:female chimerism, when I accidentally used untreated FBS to make my washing buffer.
I should add that there are specific times you need to add azide to prevent capping, internalization or shedding of your antigen of interest. Historically, it was essential to add azide because virtually all staining protocols used fluroescently-conjugated secondary antibodies to visualize the cells, and the secondary antibodies cross linked the primary antibodies, which often led to capping, internalization or shedding. This is not necessary for most primary antibodies if they are directly conjugated. However, there are some antigens, such as the receptor tyrosine kinases c-fms, c-kit and flt-3 that will cap, internalize and/or shed on direct binding. For those stainings, you must include azide, which inhibits metabolic processes, and you must do the staining and washes at 4oC.
Dear Ahmed Al-Omar ,
We usually use PBS+2% BSA in washing steps of flowcytomtry, sometimes we make wash buffer with 5% FBS (Heat activated) in PBS. We never used non-heat activated serum yet. But as Gary Lee Gilmore suggesting is right. your cell will be lysed due to the presence of activated complement complex.
Sodium azide is being a necessary compound of wash buffer in certain assays but not all. Rather it works as anti-fungal agants in your buffers as it tends to develop fungal growth due to presence of serum sooner than other buffers.
If you prepare WB regularly in lab (like every week) i dont think Azide is needed.
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