Flow Cytometry General Reagents

We offer a range of buffers suitable for use in cell surface and intracellular antibody staining.

Staining Buffer — suitable for all staining protocols.

This staining buffer is optimized for staining both live and fixed cells and is compatible with both conjugated and unconjugated antibodies. It can also be used as an antibody dilution medium and a wash buffer. Bovine calf serum is included to decrease background staining, and sodium azide is present to help reduce receptor capping.

Leucoperm — cell permeabilization reagent for intracellular staining.

Leucoperm reagent allows intracellular antigen analysis with the same ease as surface antigens. It enables antibody staining of proteins such as cytoplasmic enzymes, oncoproteins, cytokines, and other cytoplasmic proteins. Leucoperm fixes and permeabilizes cells in suspension while retaining cell morphology.

Fig. 1. Intracellular staining using Leucoperm permeabilization reagent. Rat peritoneal macrophages were fixed and permeabilized with Leucoperm (BUF09) and stained with Anti-Rat CD11b A488 (MCA275A488) and Anti-Mouse IgG1 A700 Isotype Control (​MCA1209A700) (Figure A) or with Anti-Rat CD11b A488 (MCA275A488) and Anti-Rat CD68 A700 (MCA341A700) (Figure B).

Fixation Buffer — paraformaldehyde containing cell fixative.

The cell fixation buffer is in a ready-to-use format, designed to maintain cellular integrity by stabilizing both surface and intracellular markers. It is particularly suitable for use prior to cell permeabilization in intracellular staining protocols or for fixing cells that cannot be immediately acquired on a flow cytometer.

Fig. 2. CD4 StarBright™ Blue (SBB)675 staining with and without cell fixation. Human peripheral blood was stained with Anti-Human CD4 SBB675 (MCA1267SBB675) and acquired fresh or after fixation with Fixation Buffer (BUF071).

Erythrolyse Red Blood Cell Lysing Buffer — ready to use 10x solution.

When analyzing whole blood by flow cytometry, removal of red blood cells is recommended if they are not required for analysis. Their presence can obstruct the cytometer, complicate gating strategies, and diminish signal clarity from target cell populations. Effective removal of red blood cells enables more accurate analysis of white blood cells, thereby enhancing overall data quality. Use the Erythrolyse Red Blood Cell Lysing Buffer to effectively lyse red blood cells from multiple species.

Activation reagents are used to stimulate cells to assess their functional responses, such as cytokine production, surface marker expression, and cell proliferation. This helps determine how cells behave under specific conditions or in response to treatments.

Cell Stimulation Reagent (with Brefeldin A) — containing phorbol 12-myristate-13-acetate (PMA) and ionomycin.

Cells are activated using optimal concentrations of PMA via protein kinase C, along with the calcium ionophore ionomycin. The addition of Brefeldin A inhibits transport from the endoplasmic reticulum to the Golgi apparatus, leading to the intracellular accumulation of cytokines and thereby enhancing their detection by intracellular fluorescent staining.

Fig. 3. IL-2 expression following cell stimulation. Human peripheral blood was incubated with or without the presence of Cell Stimulation Reagent containing Brefeldin A (BUF077A) for 5 hours. Cells were stained with Anti-Human CD3 A700 (MCA463A700) and Anti-Human IL-2 FITC (MCA1553F). 

We offer two commonly used protein transport inhibitors that can be used to enhance intracellular cytokine staining. As cytokines and chemokines are usually secreted, the use of these inhibitors traps the proteins within the cell, leading to a build-up of cytokines in the Golgi apparatus and endoplasmic reticulum.

Monensin — available as a 1000x ready-to-use solution.

This inhibitor acts by interfering with ion transport across cell membranes. Interfering with the Na+/H+ exchange in the Golgi apparatus halts protein transport. Monensin does not completely block all cytokine secretion. In those instances, it should be used in combination with Brefeldin A.

Brefeldin A — available as a 1000x ready-to-use solution.

This inhibitor prevents vesicle formation and protein trafficking. It redistributes proteins from the Golgi apparatus to the endoplasmic reticulum, resulting in the accumulation of intracellular cytokines.

These are used to prevent nonspecific binding of antibodies to Fc receptors found on several immune cell types, including monocytes, macrophages, dendritic cells, and B cells. Without blocking, antibodies can bind to these receptors through their constant Fc domain, leading to false-positive signals and misleading data.

Mouse Seroblock FcR — for blocking Fc receptors on mouse cells.

This reagent is a rat-derived antibody targeting mouse FcγRII (CD32) and FCγRIII (CD16), which are receptors that interact with the Fc regions of IgG molecules. The use of Mouse Seroblock FcR blocks these receptor sites so only antigen-specific binding is observed in experiments.

Fig. 4. Blocking with Mouse Seroblock FcR reduces the signal from isotype staining. Mouse bone marrow cells were unstained (Figure A), stained with Anti-Rat IgG2b PE Isotype Control (MCA6006PE) (Figure B), or blocked with Mouse Seroblock FcR (BUF041) and stained with Anti-Rat IgG2b PE Isotype Control (MCA6006PE) (Figure C). 

Human Seroblock — for blocking Fc receptors on human cells.

This reagent blocks Fc receptor binding to antigen-specific antibodies on human samples, reducing nonspecific signals for clearer, more specific staining.

Fig. 5. Blocking with Human Seroblock reduces the signal from isotype staining. Mouse THP-1 cells were stained with FITC-labeled Mouse Anti-Human CD11a in blue (MCA1848) or Mouse IgG2a Negative Control:FITC in red (MCA929F) in the absence (A) and presence (B) of Human Seroblock (BUF070A).

Excluding dead cells in flow cytometry is critical to prevent false positives, as dead cells show high autofluorescence and nonspecific antibody binding. Our easy-to-use viability assays accurately distinguish live from dead cells in fresh or fixed samples and come in various excitation and emission wavelengths for compatibility with most multicolor flow cytometry applications.

DNA binding dyes — used when cell fixation is not required.

These dyes are straightforward to use and are simply added five minutes before cell acquisition, without any washing steps required. The available options include Propidium Iodide, 7-AAD, and DAPI.

Fig. 6. Jurkat cells stained with DAPI. Plot shows unstained cells (grey) and cells stained with DAPI (1351303), showing live (open histogram) and dead (solid histogram) cell populations. Data were acquired on a ZE5 Cell Analyzer.

VivaFix Fixable Cell Viability Dyes — protein binding dyes that are suitable for fixation.

The VivaFix range is ideal to use in intracellular staining protocols or when cells require fixation for later acquisition.

Fig. 7. Jurkat cells stained with VivaFix Cell Viability Assays. Plots show unstained cells (grey) and cells stained with VivaFix (1351115 or 1351116), showing live (open histogram) and dead (solid histogram) cell populations. Data were acquired on a ZE5 Cell Analyzer.

Cell proliferation is a tightly regulated process that may change in response to stimuli like drug treatments. Dyes and BrdU antibodies are used to measure proliferation by flow cytometry, with several excitation and emission wavelengths available.

CytoTrack and CFDA-SE — fluorescent nontoxic cytoplasmic dyes.

These dyes are cell-permeable and, once internalized, are retained within cells where they become strongly fluorescent. As cells divide, the dye concentration is reduced by half with each division, enabling the tracking of cell proliferation.

Fig. 8. Resolution of ten cell divisions using CytoTrack Green 511/525 (1351203).

BrdU antibodies — to detect proliferation of BrdU-treated cells.

BrdU is a thymidine analog that incorporates into the DNA of proliferating cells in place of thymidine when present in culture media. Fluorescently labeled BrdU antibodies detect this incorporation. Used with DNA stains like Hoechst 33342, propidium iodide, or DAPI, BrdU allows the determination of the proportion of cells in S-phase.

Fig. 9. Cell proliferation detected using anti-BrdU antibodies. BrdU-labeled human lymphoma cells were stained with Anti-BrdU Antibody (MCA2483) and detected using DyLight 650 secondary antibody (STAR117D650). Hoechst 33342 (1351304) was used to stain total DNA.