Flow Cytometry Protocols

Different cell types vary in their phosphatidylserine (PS) content, along with the amount of PS exposure on the cell surface after cell death. This protocol is a guideline for getting started, however it may be necessary to adjust the concentration of the Annexin V-FITC. - [Read Annexin V Protocol for Flow Cytometry]

Annexin V Staining Flow Cytometry Protocol  - a general protocol for staining cell for cytometry analysis. BD PharMingen. - [Read Annexin V Staining Flow Cytometry Protocol]

A sensitive method for the detection of apoptosis by single laser flow cytometry. Methodology includes: Staining for detection of apoptosis, Direct Staining Procedure, Indirect Staining Procedure, Protocol for the use of actinomycin D (AD) on samples that were stained with 7-AAD for apoptosis and fixed in formaldehyde. - [Read Apoptosis Detection Protocol By Single Laser Flow Cytometry]

Basic Method for Direct Immunofluorescence Labeling. Iowa State University, Flow Cytometry Facility. - [Read Basic Method for Direct Immunofluorescence Labeling]

Protocol for calcium flux analysis. Includes: Reagent Preparation; Sample Preparation; Cell Surface Staining; Instrument Set up; Optimizing the Instrument Settings; Recording Data to Apply Compensation; Recording Experimental Data. - [Read Calcium Flux Analysis Protocol for Flow Cytometry]

This assay is based on carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. Includes: Working Dilution of FMK-peptide inhibitors; 1X Working Dilution Wash Buffer; Protocol for Flow Cytometry. - [Read CaspaTag Caspase Activity Protocol]

Protocol uses specific antibodies coupled to one of four fluorochromes: fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), peridinin chlorophyll-a (PcP), and allophycocyanin (APC). These fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Protocol for DNA analysis by flow cytometry. - [Read DNA Analysis by Flow Cytometry Protocol]

Protocol for estimating the number of CD38 molecules on the CD8+ T lymphocytes of HIV-infected individuals. Includes: RECOMMENDATION OF VENDOR FOR PE-CD38 AND PE-CD4; VALIDATION OF LOGARITHMIC AMPLIFIER LINEARITY AND SENSITIVITY; CONSERVATION OF THE LEVEL OF CD4 ANTIGEN EXPRESSION ON CD4+ LYMPHOCYTES AND ITS USE AS A BIOLOGIC STANDARD FOR FLOW CYTOMETER INSTRUMENT CHARACTERIZATION; DETERMINATION OF THE NUMBER OF CD38 MOLECULES PER CD8+ CELL; etc.. - [Read Estimating the Number of CD38 Molecules on the CD8+ T Lymphocytes of HIV-Infected Individuals]

Protocols for FASC analysis of cell cycle using BrdU and PI. Includes: Labeling of Cells with BrdU; Collecting the Cells; Processing the Cells. - [Read FASC Analysis of Cell Cycle using BrdU and PI Protocol]

FIXATION and DNA Staining for Cell Cycle Analysis Protocol. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells.  Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix.  Ribonuclease-A is used to eliminate the staining of double-stranded RNA. - [Read FIXATION and DNA Staining for Cell Cycle Analysis]

Flow cytometric analysis of gram-negative bacterial cells. Includes: Staining Conditions and FACSort Settings; Light Scatter Resolution on the BD FACSort; Bacterial Cell Resolution on the BD FACSort. - [Read Flow Cytometric Analysis of Gram-Negative Bacterial Cells]

Flow cytometric determination of leukocyte surface antigens in whole blood. Quantitation of cell surface antigens in whole blood with the flow cytometer is very simple and requires:
1. Blood collection; 2. Addition of antibody; 3. Calibration of the flow cytometer; 4. Making measurements. - [Read Flow cytometric determination of leukocyte surface antigens in whole blood]

Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules. Includes: Immunofluorescence Staining; Flow Cytometry Analysis. - [Read Flow Cytometry Analysis Protocol]

Protocol for the immunofluorescent staining of rat thymocytes. - [Read Immunofluorescent Staining of Rat Thymocytes Protocol]

Protocol for in vitro class switching staining for flow cytometry. - [Read In Vitro Class Switching Staining for Flow Cytometry Protocol]

An Indirect Immunofluorescence Labeling Protocol for flow cytometry. Iowa State University, Flow Cytometry Facility - [Read Indirect Immunofluorescence Labeling Protocol]

The more commonly available single-laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single-laser machines are used. This protocol describes the labeling of cells and analysis of conjugates with either dual-laser or single laser flow cytometers. - [Read Measurement of Intercellular Conjugates by Flow Cytometry Protocol]

Protocol for membrane potential analysis by flow cytometry. Includes: Preparation of the dyes; Loading of the dyes; Flow Cytometric Analysis. - [Read Membrane Potential Analysis by Flow Cytometry Protoocol]

Provides an overview of terminology and echniques unique to flow ytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation-oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry. - [Read Overview of Flow Cytometry]

Paraformaldehyde Fixation of Cells protocol. This fixation method is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens.  It will stabilize the light scatter and labeling for up to a week in most instances, allowing you to be more flexible in scheduling cytometer time.  Furthermore, it inactivates most biohazardous agents, so it is important from a safety standpoint as well. Iowa University. - [Read Paraformaldehyde Fixation of Cells]

Protocol for PARP PE. - [Read PARP PE Protocol]

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Protocol for purification of Lin-, c-kit+, Sca-1+ bone marrow cells for Culture, Flow Cytometry, or Transplantaion. Includes: Antibodies for Lineage Depletion; Harvest Marrow; Lineage Depletion; Cell Sorting; Viral Transduction; Transplant; CFU assays; GFP and cell surface marker immunostaining. - [Read Purification of Lin-, c-kit+, Sca-1+ Bone Marrow Cells for Culture, Flow Cytometry, or Transplantaio]

Protocol for sodium and potassium analysis by flow cytometry. Includes: Preparation of the dyes; Loading of the dyes; Flow Cytometric Analysis. - [Read Sodium and Potassium Analysis by Flow Cytometry Protocol]