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Flow Cytometry Protocols

Sample Preparation

 

 

Staining cells with a Lyse/No-Wash protocol


Direct Immunofluorescence Staining of Whole Blood Using a Lyse/No-Wash Procedure

Find procedures for whole blood specimen collection and preparation, including step-by-step instructions for lysing and staining. Use this method to detect cells bearing specific membrane antigens.

 

BD™ Phosflow Protocols for Mouse Splenocytes or Thymocytes

Explore four protocols using different methods (detergent method, mild or harsh alcohol method or harsh detergent method) for the activation of cell signaling molecules in mouse splenocytes or thymocytes.

 

 

Staining Mouse and Rat Leukocytes


Immunofluorescent Staining of Mouse and Rat Leukocytes

Find instructions for immunofluorescent staining procedure and staining tips for mouse and rat leukocytes.

 

 

Indirect Staining Mononuclear Cells


Indirect Immunofluorescence Staining of Mononuclear Cells

The indirect method is used to enhance the fluorescence signal and also to facilitate multicolor staining of human cells when direct conjugated reagents are not available. Find the procedure for specimen collection, preparation and staining of mononuclear cells.   
  

BD™ Phosflow Protocols for Human PBMCs

Explore four protocols using different methods (detergent method, mild or harsh alcohol method or harsh detergent method) for the activation of cell signaling molecules in  human peripheral blood mononuclear cells.

 

BD™ Phosflow Protocols for Human Whole Blood Samples

Explore four protocols using different methods (detergent method, mild or harsh alcohol method or harsh detergent method) for the activation of cell signaling molecules in  human whole blood samples.

 

 

Reducing non-specific staining with Fc Block


Uses of Fc Block

Learn how antibody binding to Fc receptor can cause background staining and how to use the BD Fc Block™ Reagents in immunophenotyping of mouse or rat leucocytes..   

  

Protocol for Direct Immunofluorescence Staining of Mononuclear Cells

Find the procedure for specimen collection, preparation and direct immunofluorescence staining of mononuclear cells..

 

Cell Surface Staining of Human PBMCs and Suspension Cell Lines

Find a step-by-step procedure for cell surface staining of human peripheral blood mononuclear cells and cell lines.

 

Phosflow Protocol for Adherent Cells

Find the optimal protocol for detecting intracellular signaling molecules from adherent cells using BD Phosflow™ Reagents.

 

Intracellular Flow and Phosflow FAQ

Find answers to most commonly asked questions on intracellular flow cytometry and use of BD Phosflow™ Reagents..

 

TCR Stimulation and 96 Well BD Phosflow Protocol

Find the complete procedure for TCR stimulation of peripheral whole blood cells using BD Phosflow buffers.

 

 

Choosing the Right Fix Perm Kits


Choosing the Right Fix Perm Kits Cell Fixation/Permeabilization Kits for Intracellular Cytokine Analysis

Explore three cell fixation/permeabilization kits to simplify the preparation of cells for intracellular staining of cytokines.

 

   

Controls


Isotype Control Antibody Selection Guide

The selection of an appropriate isotype control is essential for every flow cytometry experiment. Find procedures for determining the isotype of your primary antibody, for searching for your isotype control and for using your isotype control.

 

 

Instrument Setup


Procedure for Manual Setting of Compensation for Multi-Color Flow Cytometric Analysis

Find detailed information on electronic compensation for fluorochrome spectral overlap during flow cytometric analysis of multi-color immunofluorescence staining. We also offer a detailed procedure for setting compensation for multi-color flow cytometric analysis.

 

 

Troubleshooting
 

The Escapee Phenomenon

Escapees are aggregates of cells (including positively labeled cells) that are excluded from the flow cytometric analysis gate, thus altering the results of the analysis. See recommendations to decrease the number of escapees.

 

 

Intracellular Cytokine Staining Protocol


Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis

Get information on stimulation of cells, appropriate cultures for generating human, mouse and rat cytokine producing cells and describes a protocol for multicolor staining of intracellular cytokines and cell surface antigens. It also offers an alternative protocol for the activation and intracellular staining of whole blood.


Cytokine Stimulation and 96 Well BD Phosflow

Explore the complete procedure for cytokine stimulation of peripheral whole blood cells using BD Phosflow buffers.  

 

 

Immune Cell Activation


Activation of Immune Cells for Cytokine Production

Explore ways to activate human  and mouse T cells. You can also find information on cultures for generation human, mouse or rat cytokine-producing cells.


Cell Fixation/Permeabilization Kits for Intracellular Cytokine Analysis

Find protocols for fixing and permeabilizing cells using various BD Biosciences products, such as BD Cytofix™ Kits, BD Cytoperm™ Kits, BD GolgiStop™ and BD GolgiPlug™ Kits. 

 

 

 

Downloadable BD Biosciences Protocols

 

Simultaneous Measurement of Mouse Foxp3 and Ki-67 in Cultured Splenocytes
 
Simultaneous Measurement of Human FoxP3 and Ki-67 in Cultured PBMCs
 
Generation of Human IL-17–Producing Cells (Th17-like Cells) In Vitro Live Cell Discrimination and Measurement of Phosphorylation and Cell Surface Markers in Thawed and Activated Human PBMCs
 
Freezing and Thawing of Peripheral Blood Mononuclear Cells (PBMCs)
 
Preparation of Murine Splenocytes
 
Tissue Preparation for Surface Antigen Staining
 
Bulk Erythrocyte Lysing with Ammonium Chloride for Flow Cytometry
 
Immunophenotyping