This reduces the chance of heterophilic antibody interference. In most antibody assays, protein is used to prevent these interactions. Heterophilic antibody interference causes both false positives and false negatives.

Also, if you just put 10mL of FBS (or FCS) in 500mL sterile 1X D-PBS, as long as you're using good sterile technique, this solution doesn't require the addition of sodium azide.

Good luck.

Blocking non-specific background staining by antibody.

The reason is the one above mentioned by Ayesha. You could use PBS+BSA instead of you want to avoid FCS.

Adding a bit of protein (generally FBS or BSA) can reduce non-specific binding by your antibody as well as reduce cell clumping with some cell lines.

All of the above and cell viability.

This reduces the chance of heterophilic antibody interference. In most antibody assays, protein is used to prevent these interactions. Heterophilic antibody interference causes both false positives and false negatives.

Also, if you just put 10mL of FBS (or FCS) in 500mL sterile 1X D-PBS, as long as you're using good sterile technique, this solution doesn't require the addition of sodium azide.

Good luck.

FBS highly improves cell survival after the sorting, specially if you deal with fragile cells like neurons or stem cells.

Use of FBS/FCS or other serum in any immunostaining procedure (IF,WB,IP,Flow Cyt, IHC-f/p, ICC etc) is just for avoiding non specific binding by blocking other sites of proteins, so it reduces background noise. This serum also helps to stabilize all the proteins in funtions i.e. incease the viability of cells. FCS contains a lots of different proteins which bind to non-specifically all the proteins loosely so it makes binding of antibody to the specific epitope little bit difficult but more specific due to high affinity between the anibody binding site and antibody it replaces the serum proteins at the specific epitope but not able to replace the serum proteins at other site because of similar affinities.

All of the above responders are correct. I would add that it may be useful to add 2 mM EDTA as well to prevent cells from interacting and cause a high forward scatter as doublets or bigger clusters.

The use of EDTA over prolonged incubations may lead to a decrease in cellular viability. Although it is useful to avoid cell clumps, it should be washed away after the first staining steps. Also, use it carefully if highly cationinc buffers like those for Annexin V staining are going to be employed.

FBS increases the cell viability.

As already stated the addition of protein (FBS or BSA) helps reduce non-specific antibody interactions and cell aggregation. I believe they also help with maintaining osmolarity of the buffer and overall cell viability.

Azide is a metabolic inhibitor and is added for several reasons :

1. prevent bacterial/fungal growth

2. allows storage of buffer and staining at room temperature

3. inhibits capping, antigen internalisation and shedding of surface antigens

4. inhibits antibody internalisation

Avoid azide supplemented media if subsequent cell function is required, in which case filter PBS supplemented with 0.1-2% FCS(or BSA - generally has significantly lower associated autofluorescence ) and use cold for staining steps to mitigate 3 and 4

Use of FCS or BSA in in FACS buffer reduces autofluorescemce caused by non specific biding by antibodies which may falsely increase the MFI of a channel in flow cytometery

Using BSA of FBS in FACS buffer might help to cover non/speciffic binding, similarly as using 5% BSA for blocking in western blots.

Hi anyhow while staining you would be using PBS-T.

But acquiring, you may use sheath buffer, which has proteins (BSA), but you can omit using sheath buffer. If using live cells its better to use sheath buffer.

If you use, fixed stained cells then PBS should do.

I fully agree with Martin Waterfall's answer!

Use of FCS or any other serum or even BSA in FACS buffer is just to provide stringency to Ab to avoid non-specific binding other than its epitope. Otherwise, one may get false-positive signal which may lead to overestimation.

When staining for surface and intracellular molecules, many a times, we never got a signal from the surface antibody which could be due to antigen capping?!! I am not sure but addition of azide (0.6%) in the wash solutions stopped this. Otherwise the addition of azide may be skipped if cells are to be sorted and cultured for downstream applications.

In general all immunological methods use protein in the staining buffers to prevent/reduce unspecific adherence of antibodies to a) the plastic and b) to cells. You can use any protein (e.g. 1%BSA as used in ELISA, 1% non fat milk powder, 1% soja protein powder or FBS). Labelling of cells for flow cytometry should always be performed with cold buffers because the cells will keep round, will not make cellular extensions (e.g. filopodes/mikrospikes) and will not internalize or up-regulate cell surface proteins. Thus and due to the relative short time of the staining procedure for flow cytometric approaches (e.g. 1h primary antibody, 30 min fluorochrome labelled secondary antibody) there is no effect on cell survival, cell activation etc.

The use of FBS reduces unspecific binding, thus giving a better signal to noise ratio as a result. However specific IgG or IgM isotype control should always be added to set the basal level of fluorescence during the analysis, especially if the result is expressed as MFI/RFI.

FBS or BSA deals with unspecific binding, maintains osmolarity, reduces cell aggregation and thus finally decrease the MFI of a channel in flow cytometery.

FBS or BSA [or casein or any other protein] blocks non-specific binding of proteins to surfaces, be they glass or plastic. Without it, cell surface proteins may stick, and shear off the surface of the cell, reducing viability. When you are using fixed cells, this is no longer a problem.

Use of azide in flow cytometry is actually to prevent capping of antigens, which was very common when staining is accomplished with 2o antibodies. Many antigens cap - particularly surface immunoglobulin on B lymphocytes - but some do not. You need to empirically determine if your signal is lost in buffer without azide.

All important points made above. I would add that I would just get into the habit of keeping all of your buffers at 4˚C (no need to buy commercial buffers PBS/FBS or BSA/azide just as good) and staining on ice, keeping antibodies and buffers on ice throughout. No harder and means you are doing all you can to clean up your plots. Bit belts and braces (ie 4˚C and azide) but you will get beautiful results. The only tails on your histograms will be real not artefact!!

As teh oterhs have stated, it presvnets non-specific binding to cells and for primary cells (e.g. PBMCs) keep them from clumping and sticking to the palstic. You coudl use 1% BSA instead.

0.1-0.2% FBS is required to keep cells alive and metabolically functional outside CO2 incubator when you're working with them for long in ice till flow cytometry analysis. Unless you are using 2% formaldehyde to fix the cells and surface markers. BD bioscience has many good protocols for flow process to start with..

I actually do a lot of human hematopoietic stem cell analysis with directly conjugated monoclonal antibodies without using azide in my working buffer - CD45,CD34, CD38, CD133, CD90 - these are all stable and do not cap. Neither do CD4 or CD8. However, staining of certain antigens - CD115, CD135, CD3 do cap and require working buffer with azide and keeping the tubes on ice or in the refrigerator to see the antigens. So you either have to test your antigen/antibody of choice to see if it's stable, or to be safe, you can do all samples on ice with azide. It's all a matter of what works for your system.

We do get some non specific binding when we look for light chains on B-lymphocytes. Any suggestions of protocols how to prevent this using FBS or BSA? 

FBS is not compulsory, you can substitute with BSA. It prevents non-specific binding maintaining buffer Add to dictionary as well as well as prevent damage to cells. You can add azide if you want to store the buffer, but if you don't want to store it azide is not required. Always use ice chilled buffer.

This is great information, thanks guys for highlighting this aspect of cell line maintenance.