Clone selection...

Ensure antibody clone / manufacturer differences are tested when selecting antibodies for a panel.

a)

b)

(a) Two clones were available for the transcription factor PU.1 (found in some myeloid cell subsets). Both are on the same fluorophore, but only clone 7C6B05 was able to resolve a separate population. A titration was preformed, but the population was never discernible by clone 7C6B05.

(b) Here the same clone from two different companies on two similar fluorophores was evaluated on the same PBMC donor. You can see company A conjugated-antibody was able to resolve a CD3+ CRTH2+ population, however company B’s conjugated antibody was not. Increasing the concentration did not help and simply resulted in more spread in the negative.

Viability titration

Benefits of titrating not only antibodies but viability dyes. 

(above) Titration revealing populations that are unresolvable with an abundance of antibody due to non-specific binding.

(below) Often overlooked, titrating the amine-reactive viability markers can result in tighter negative populations due to minimised unbound fluorophores and baseline restoration, which has less of an effect on other important markers even in the negative (live) population.  

Annotate your experiment before or during data acquisition

The MIFlowCyt Standard (minimum information necessary for flow cytometry experiments) outlines the minumum information required to report the experimental details of flow cytometry experiments. 
Allocate time to annotate appropriate metadata, labelling the fluorochromes & antibodies used, as well as other experimental information to allow re-interpretation of the results in the future. 

Identify dead cells and debris

Dead cells can lead to inaccurate frequencies and can increase non-specific staining. Identifying them with one or more of the many viability/apoptotic markers can clean up your resultant data significantly making interpretation significantly easier. DAPI, 7-AAD, PI, TO-PRO-3, SYTOX green, DRAQ7, Calcein-AM and amine reactive dyes are examples that can be used. 

Centrifuging fluorescent antibodies prior to using them may remove aggregates resulting in cleaner data

Sometimes you may notice a number of events above the obvious positive population, shown below. On occasion these may be fluorescent molecule aggregations that are super bright. 1 trick (and I hope to upload a before and after example when I dig it up) is to either individually centrifuge antibodies before using them, or centrifuging the cocktail before adding to cells in order to remove fluorescent aggregates. In a micro-centrifuge, 10000-12000g, chilled, 5 minutes can be used to pellet aggregates, followed by pipetting the supernatant.

Ensure FSC-A, FSC-H, FSC-W & SSC-A, SSC-H, SSC-W are collected

The digital instruments at WRHFlow allow the collection of additional parameters that can be used to distinguish cell doublets and aggregates of cells from single cells before enumeration.

Avoid clumps in your sample - filter them through a mesh filter

Flow cytometry is a single cell analysis platform (for the most part). Clumping cells can ruin data acquisition as they may clog the narrow orifice the sample needs to travel through to enter the flow cell resulting in incorrect signals being assigned to events due to the way the instruments works. Filtering your samples before running on the cytometer can assist in minimising this occurring. Other things to try might include adding 2mM EDTA into your buffer, enriching cells from debri/dead cells/free DNA before acquisition, keeping your sample cold, and diluting your sample. 

Good practice includes having a time vs. fluorescent parameter (off the last laser used) to monitor your acquisition to spot problems and fix them before finishing running your sample.