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Tips and tricks to optimize Flow Cytometry

Flow Cytometry protocol optimization 
Dr. Camilo Moncada, Ph.D. - Rockland Immunochemicals

Flow cytometry (FCM)  is a powerful quantitative technique that provides information regarding the biological properties of cell populations based on the expression of one (or several) intracellular and cell surface antigens.

A number of methods and strategies have been developed to get the most out of flow cytometry. Here, Dr. Moncada (Director of QC at Rockland Immunochemicals) shares some of those that he, and his team, have found useful in their lab.

To learn in more detail about how to improve the performance of flow cytometry experiments, go to Rockland's detailed protocol description including a comprehensive list of reagents, or contact tebu-bio's team of experts to assist you in the selection and the use of the your FCM reagents.

About Dr Moncada

Camilo Moncada, Ph.D. is Director of QC at Rockland Immunochemicals, a leading company in antibody development.

Dr. Moncada performed research studies for more than 10 years and was an active participant in several diverse projects resulting in publications in the areas of immunology, parasitology, cancer and lung disease. At Rockland, he applies his expertise on molecular and cellular biology, biochemistry and proteomics for antibody development and validation.

1. Choosing the right controls for flow cytometry

Always remember to include negative controls of the same isotype as the labeled antibody so that you can determine the extent of background signal in your experiments.

FCM Mouse IgG1 isotype Contrl PE 010-0840 Rockland tebu-bio

This is true when staining with a labeled primary antibody only, or when using a combination of primary antibodies and phycoeryhtrin (PE) labeled secondary antibodies (ex. Mouse IgG1 isotype control PE cat. nr 010-0840).

For best results remember to always include a sample of unstained cells (incubated in parallel with your stained samples) so that you can control for background derived from auto-fluorescence.

Whenever possible include cells known to express the antigen of interest as well as cells known to lack the same protein since they will help to determine the specificity of the antibodies used.

2. Reducing noise from your fluorescent signal

A critical step during optimization of flow cytometry experiments is reagent (antibody) titration. For optimal results you must determine the minimum amount of antibody required to achieve antigen binding saturation. This will help you improve the specificity and intensity of your fluorescent signal while minimizing background.
If you are simultaneously staining with multiple colors, this titration step will also allow you to sense unexpected cross-reactivity between FCM validated primary and labeled secondary antibodies.

3. Enhance permeabilisation and fixation to improve detection of intracellular proteins

Intracellular targets entail a more stimulating detection since they have additional requirements that go beyond antibody specificity, including the ability to successfully cross the permeable cell membrane.
Optimization of cell fixation and permeabilization is a critical parameter that needs to be empirically determined in order to balance the integrity of the intracellular structures with cell permeability.
Always use freshly prepared solutions of high purity paraformaldehyde for fixation and initially try mild detergents (i.e., Tween 20) for permeabilization.
If your fixation and permeabilization steps need further improvement, try increasing the formaldehyde concentration gradually up to 4% or try ethanol or methanol and using other detergents including Triton X-100, or saponin without fixation or alcohol fixation.Anti-CD47 PE in FCM

4. Maintaining fluorescent signal at high intensity 

When performing staining of cell surface markers always consider the possibility that extracellular antigens may be internalized upon antibody binding. This naturally occurring phenomenon can have a significantly negative effect on the intensity of your fluorescent signal and can be prevented by the following steps:
  • Working with aggregate-free antibody solutions
  • Keeping your samples ice-cold or refrigerated
  • Including sodium azide in the staining buffer so that cellular metabolic activity is down-regulated during staining.

Also consider using monovalent antibodies in the form of F(ab) fragments.

5. Stain dead cells to obtain meaningful data from viable cells

Since dead cells can bind non-specifically to any antibody, it is imperative to keep those out of the analysis. The best way to do this is to use a fluorescent dye that will pass through damaged plasma membranes and thus stain dead cells.
In most cases, especially when performing staining of intracellular targets, it is advisable to use dyes that covalently bind and stain non-viable cells so that they won't leak out once the cell is permeabilised

Featureds intracellular target primary antibodies for FCM

Anti-Lysine Acetylated (AcK) Antibody
Stem Cell Antibodies
Anti-NOTCH 2 (Cleaved N terminal) (Human specific) Antibody
Anti-Telomerase catalytic subunit Antibody
Transcription Factor Antibodies
Anti-CREB-1 (p43) Antibody
Anti-NFKB p50 (NFKB1) Antibody
Phospho Specific Antibodies
Anti-AKT pS473 Antibody AKT1 Antibody PE Conjugated 200-308-J34 in FCM
Anti-Aurora B pT232 Antibody
Anti-DNA PKcs pT 2609 Antibody
Neuroscience Antibodies
Anti-Gli-3 Antibody
Anti-COX-2 Antibody
Cancer & cell signaling
Anti-BrdU Antibody 
Anti-AKT2 Antibody PE conjugated
Anti-NOTCH 2 Antibody (Cleaved N terminal) (Human specific) Antibody
Hematology / Immunology studies
Anti-CD19 Antibody APC conjugated Anti-CD8 antibody APC in FCM
Anti-CD4 Antibody Fluorescein conjugated

 Featured isotype controls and secondaries for Flow Cytometry

tebu-bio would like to thank Dana, Camillo and Carl from Rockland for preparing this document.