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8 Sample Prep Mistakes To Avoid

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Flow cytometry training often focuses primarily on how the instrument works, how to establish good compensation and of course, how to do the data analysis to get those publishable results.

But what about preparing the sample in the first place?

Does anyone know why the protocol calls for 5% normal rat serum or why is there BSA in the staining buffer? 

To generate successful flow cytometry data, it all starts with good sample preparation. A lot of scientists make sample prep mistakes that can cost them their experiment. Here are 8 mistakes that you should be careful to avoid:

1. Treating all cells the same.

Depending on the origins of the sample, it is critical to review how to get the cells into a single cell suspension. It is easy with blood and non-adherent cells. However, if using an adherent cell line, or a solid tissue, dissociation methods need to be robust enough to disassociate the tissue but gentle enough to not destroy the cells. A good resource to review is the Worthington Tissue dissociation guide. 

2. Failing to minimize non-specific binding.

Non-specific binding and Fc receptor binding must be addressed at the beginning of the assay, not the end. To minimize non-specific binding, consider adding some normal serum. For example, if the assay is using mouse-anti-human antibodies, block with 10% normal mouse serum for 10 minutes before starting the staining process. One caveat for this is that if using an amine-reactive dye, incubate with the serum after labeling with the amine reactive dye.

3. Forgetting to titrate.

A second way to minimize NSB is to titrate the antibodies. This ensures that the correct amount of antibody is being used. If too much Ab is used, this increases NSB and thus background. If too little Ab is used, this reduces the signal, and reduces the ability to detect the cells of interest.

4. Not counting cells at both the beginning and the end.

Every time the cells are spun down and washed, there are losses. Before going to the cytometer, it is good to know how many cells are there - this will confirm that there are sufficient cells to measure the outcome. If, for example, the population being measured is 0.01%, and there are only 100,000 cells in the tube, that mean on 10 cells (maximum) will be measured.

5. Ignoring tube choice.

Depending on the assay, the tubes could make a difference. For example, if performing a fix/perm assay, cells will not pellet well. Using a silanized tube will help the cells pellet. However, the cells will be loosely adhered to the tube, so removing the supernatant must be done with care.

6. Treating all buffers the same.

There are as many different recopies for staining buffer as there are labs. Some groups use BSA as a protein, others use FBS. It is important to remember that FBS can have different compounds that can negatively impact different cell types. For this reason, if using FBS, make sure that each lot is tested and not activating the cells of interest. Better yet, consider using BSA for the protein.

7. Forgetting to filter the cells.

Flow cytometry requires single cell suspensions - so to ensure that the cells are not clumping, filter them before going to the cytometer. If in doubt, check out the sample under the microscope.

8.  Not checking the centrifuge speed.

All protocols should report the centrifuge speed in xg - not RPM. Knowing how many times G that the cells should be pelleted at is critical. Since each centrifuge is different, RPM is not a good measure of how best to pellet the cells.

In conclusion, review the protocol before delving into the assay. Anything that doesn’t make sense or is unclear should be ferreted out and resolved. Good cytometry can’t be done without a good sample, and attention to detail in the area of sample prep is the key to that.