Game 4: Intracellular Staining
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When you assess intracellular markers in your assay, fluorochrome choice can have a significant impact on your results. Not all fluorochromes work well for intracellular staining, as some fluorochromes are impacted by buffers used to fix and permeabilize the cells.
In this Fluorochrome Faceoff, we pair fluorochromes that are excited off the blue laser and emit around 610 nm. We selected several different intracellular markers to evaluate and stained them with three different fluorochromes. As you can see, each dye provides good population resolution and the expected staining pattern. Please note that there are clone differences between the dyes.
Top: Two-color flow cytometric analysis of Granzyme B expression in human peripheral blood lymphocytes. Human whole blood was fixed with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD8, clone RPA-T8 (Cat. No. 561953) and BD Horizon™ RB613 Mouse Anti-Human Granzyme B, clone GB11 (Cat. No. 571117), BD Horizon™ PE-CF594 Mouse Anti-Human Granzyme B, clone GB11 (Cat. No. 562462) or BioLegend PE/Dazzle™ 594 Mouse Anti-Human Granzyme B, clone QA18A28 Reagent. The bivariate pseudocolor density plots showing the correlated expression of Granzyme B versus CD8 were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. .
Middle: Two-color flow cytometric analysis of IL-17A expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 µg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). The cells were washed then permeabilized and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 SK3 antibody (Cat. No. 555349) and either BD Horizon™ RB613 Mouse Anti-Human IL-17A, clone N49-653 (Cat. No. 571130) or BioLegend PE/Dazzle™ 594 Mouse Anti-Human IL-17A, clone BL168 Reagent. The bivariate pseudocolor density plots showing the correlated expression of IL-17A versus CD4 were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. NA indicates that the specificity/format combination was not available to us at the time of testing.
Bottom: Two-color flow cytometric analysis of Ki-67 expression by proliferating human MOLT-4 cells. Proliferating cells from the human MOLT-4 (T lymphoblastic leukemia, ATCC® CRL-1582™) cell line were permeabilized and fixed with 70% ice-cold ethanol. The cells were washed twice with BD Pharmingen™ Stain Buffer (Cat. No. 554656), stained with BD Horizon™ RB613 Mouse Anti-Ki-67, clone B56 (Cat No. 571126), BD Horizon™ PE-CF594 Mouse Anti-Ki-67, clone B56 (Cat. No. 567120) or BioLegend PE/Dazzle™ 594 Mouse Anti-Ki-67, clone 11F6 Reagent and counterstained with BD Pharmingen™ DAPI Solution (Cat. No. 564907) to stain DNA. Bivariate pseudocolor density plots showing the correlated expression of DAPI staining versus Ki-67 expression were derived from gated events with the forward and side light-scatter characteristics of MOLT-4 cells. NA indicates that the specificity/format combination was not available to us at the time of testing.
Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.
Winner’s Circle
Because all three of the dyes tested perform well for intracellular staining, we have classified them all as winners! Please note that as of July 26, 2024, StarBright™ and NovaFluor™ Dye-conjugated antibodies are currently only available against cell surface markers and are not currently conjugated to intracellular markers. They were, therefore, not included in this Fluorochrome Faceoff.
PRO TIP: In addition to fluorochrome sensitivity, different clones are also differentially impacted by treatments with different permeabilization protocols and therefore it is important to make sure you are using an antibody clone that can withstand these protocols.
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Disclaimer: Results and conclusions shown throughout the Fluorochrome Faceoff are based on experiments performed under the conditions described. Users should evaluate reagents with their specific protocols as results may vary with different experimental conditions.
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