Flow Cytometry experiment comprise of many small and crucial step, which need to be standardize and optimize for proper experiment with great results.

*Flow Cytometry Sample Preparation: Foremost requirement of flowcytometry experiment is that cells under analysis must be in a single-cell suspension and to avoid unwanted instrument clogs to produce consistent data. results. Cells for flow cytometry analysis are usually derived from different source like adherent or suspension cultures, Cryopreserved samples, Whole blood, Solid tissues. Doing all this preparation end at a homogenous single-cell suspension free of clumps and debris with density of 106-107 cell per ml suspended in a suitable staining buffer:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*Flow Cytometry Protocols: click here

*Flow-cytometric controls:click here

 

*Optimization of Flow Cytometry experiment: There are many crucial steps or tips can be use to optimise experiment for better result at the end.

How to prepare sample and maintain cell quality?

  • If possible, use freshly isolated cells rather than frozen and thawed cells.

  • Cell numbers affect the staining quality as well as the efficacy of any downstream assay. Furthermore, cells are steadily lost during the staining procedure. it is therefore imperative that enough cells are available at the start of the experiment to compensate for the inevitable losses

  • It’s better to pipette the cells gently to prepare homogenous single-cell suspensions in compare to vortexing to avoid cell disruption.

  • Remove clumps and other debris by sieving through nylon filters in final suspension.

  • For cryopreseve cells, perform the initial dilution of the thawed cells at a high serum concentration (90% FBS in culture medium) to increase viability of thawed cells.

  • When isolating cells from complex tissues, it is better to perform the steps on ice (except for the steps involving digestion enzymes) to prevent phagocytosis and cell lysis.

  • If the downstream procedure is live cell sorting, it is recommended that cells are counted after each step to ascertain viability.

 

Fixation and Permeabilization Method

  • Fixation/permeabilization reagents alter the scatter properties as well as the auto-fluorescence of cells. Therefore it is recommended to include an unstained control that has been treated with the same reagents.

  • For combined surface and intracellular staining, it is advisable to first stain for the surface markers and then fix/permeabilize as the latter can alter some antigen epitopes and affect antibody binding.

  • For the staining of secreted proteins like cytokines, a protein transport inhibitor such as Monensin or Brefeldin A should be added prior to fixation/permeabilization in order to trap the cytokines inside.

  • Binding of antibody to surface antigen can stimulate the cells and alter the expression of intracellular signalling proteins. Therefore, surface staining should always be performed after the stimulation.

  • For phosflow staining, the cells should be fixed and permeabilized immediately after the stimulation as phosphorylation is a transitory phase and quickly pass.

  • Choosing the right permeabilizing agent is extremely important – one can choose between detergents like saponin or TritonX and methanol.

    1. Saponin does not alter the surface antigen epitopes so surface staining can be done afterwards.

    2. TritonX and Tween should be avoided as they can lyse cells if incubated for long.

    3. Methanol is compatible with most intracellular antigens and cells treated with methanol can be stored at -20 to -80°C for an extended duration without loss of signal.

    4. Not all fluorochromes however can withstand methanol treatment. The commonly dyes which are methanol resistant PE, PerCP, APC, All Tandem dyes  and methanol sensitive FITC, eFlour 450, eFlour 660, Alexa Flour 647, Alex Flour 488.

 

Point during multicolor panel design:

  • Always try to pair the brightest fluorochromes with the weakest expressing antigen. In case the expression level of an antigen is unknown, it is advisable to use brighter fluorochromes.

  • Avoid spillover or spectral overlap between fluorochromes by spreading them as much as possible across the spectrum.

  • Avoid fluorochromes that can be excited by more than one laser e.g. APC-Cy7.

  • Always include suitable compensation controls especially.

  • It is also advisable to use FMO controls to gate differently stained sub-populations more accurately

 

Flow Cytometry Immuno-staining:

  • The storage and handling of the antibodies should done as recommended to avoid degradation and Fc receptor mediated aggregation.

  • Remove antibody aggregates by centrifuging at a high speed at 4°C 5 min. This step is not recommended for IgM antibodies and PE conjugates due to larger sizes.

  • Perform antibody binding reactions on ice and away from direct light.

  • If using adherent cell, do not digest with trypsin as the latter can cleave some surface antigens.

  • In case an antibody has to be diluted and then stored as aliquots, use the staining buffer for dilution. Add 0.09% sodium azide to prevent bacterial contamination.

  • Wash the cells once or twice in staining buffer after each incubation step to remove any unbound antibodies.

  • Include a viability dye in the antibody cocktail to gate out any dead cells or cell debris.

  • To prevent non-specific Fc receptor staining, add an Fc blocking step or include FBS in the staining buffer. Alternatively, include an isotype control to subtract any signal contributed by the Fc receptor staining.

  • It is always better to acquire the cells soon after staining to minimize any fluorochrome bleaching. In case the cells need to be stored, fix the cells in a suitable fixative and store at 4°C: for overnight storage 0.5% PFA I a good choice but for longer durations like several days or even weeks, the recommended fixative is ethanol.

  • Long term storage in fixative is not recommended as it can significantly increase auto-fluorescence.

  • Antibody titration is recommended to determine the correct concentration of an antibody for the optimum signal. Test different dilutions of the antibody to zero in on the lowest concentration that gives the strongest signal in positive control and the weakest signal in a negative control.

 

*Flow Cytometry Troubleshooting Guide: click here

There may be high background, or unexpected staining, or the fluorescence signal may be weak, or any of a thousand different problems. Proper controls can help eliminate a great number of possible sources of error, but many troubles will remain.

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       Article invited for

          Vol 5,Issue 3,

                  2020

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