Basic Experiment Recommendations

The first thing you need are appropriate controls. In almost ANY experiment, you will need an unstained control as we want to see what your background autofluorescence is within your cells. Only then can we see how much “extra” specific fluorescent label there is to see if you cells are positive for a protein marker, ROS probe, etc.

If you are using suspension cells (blood cells, etc) you can stain these samples and run them in a standard flow cytometry staining buffer (i.e. PBS with 2-3% BSA). If you are dissociating tissues or using cultured cells that are very adhesive, filtering will help remove clumps but since cells reassociate, it is advised to add 1mM EDTA to prevent cell clumping and clogging the cytometer. As a general staining protocol for extracellular antigens, use between 250k-1 million cells per mL in 100uL of buffer, staining with 1ug of antibody for 15-20 minutes. Ideally this is something you titrate but this gives a reasonable starting point for staining.

The second type of control you will need is single stained controls when doing a multicolour experiment which allows for calculating and compensating for spectral overlap between the fluorescent colours you are using. For example, a triple-stained sample for FITC, PE and BV421 wound need a sample stained ONLY for FITC, ONLY for PE, and ONLY for BV421. Note that this can either be live cells or (more easily and often-times better) compensation beads (see the useful links for some providers). Without these controls, you cannot do compensation and cannot run this multicolour panel. Specifically how to compensate will be covered during your standard flow cytometry training.

The last major control would be an FMO control which is necessary for >3 colour experiments for gating purposes and quality control. This is further detailed in the immunophenotyping section and won’t be reproduced here. In multicolour staining it is also advisable to use the spillover spread matrix (SSM)  to plan to use fluorophores that maximize resolution. If you have questions about the SSM matrix please contact the RCF flow cytometry specialist.

Isotype controls are controls no longer viewed as useful with the exception of intracellular staining procedures. This is covered in detail throughout the literature, one paper of which can be found here.

On the topic of specificity, the best bet is to use some combination of blocking strategies to destabilize low-affinity interactions as the highest affinity interaction is usually your antibody binding to the antigen of interest. There are many ways of doing this, but the most standard is to include serum protein or BSA at 1-2 % which prevents hydrophobic adsorbtion of the antibody to the plasma membrane in a non-specific manner. Serum protein also keeps the cells happy and alive to a better extent than shoving them in PBS and hoping they don’t apoptose before you get to the cytometer. Another thing people often to miss is blocking Fc Receptors which bind to the Fc region of antibodies and will happily thus look positive for any old antibody you are using, regardless of the presence or absence of the antigen you intend to stain for. This is critical when staining samples where you might have inflammatory cells present (macrophages, etc). If you are unsure whether you need Fc block, ask your local flow pro. There are several commercially available or home-made solutions to block this specific (but inappropriate) labeling of cells with your antibody-of-interest. The most effective is to buy anti-CD16/32, which is an antibody that sterically blocks FcRs.

For some motile cells where antibodies cross-link antigens, there can be a problem called “capping” whereby antigen is actively cleaved from the cell. This is something that people have had trouble with in lymphocytes and is easily remedied by adding 0.01 per cent sodium azide in your staining buffer. This blocks ATP dependent processes, including capping, which may impact your biological question(s), so keep this in mind before adding azide to your preps with reckless abandon.

The remaining experimental controls and suggestions for each type of assay are very application-specific as are the biological ones (i.e. activated vs. inactivated cells, timecourse, etc), fluorophore choice, autofluorescence etc. These are best determined through planning your experiment ahead of time with the help of the Research Core Facilities staff.

Useful links:

FcBlock:

Compensation beads:

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