14-10_house_marker_thin
 

FACS / Flow Cytometry Analysis


Phospho-Flow Cytometry Image

Flow cytometry combines cell biology with the study of light waves. Indeed, Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multi-parameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing properties of biological cells, including size, granularity, cell receptors, and even biological activities, such as DNA, RNA replication, and protein synthesis or suppression.

 

 

 Click here for a Flow Cytometry Overview



 

 

Rockland Flow Cytometry Analysis

Flow Cytometry Related Products




Flow Cytometry Protocol

This basic protocol focuses on the measurement of fluorescence intensity produced by fluorescent-labeled antibodies and ligands that bind specific cell-associated molecules.




Reagents Required

  • FACS Buffer: 0.5% BSA (Code # BSA-10), 0.05% Azide in PBS (sodium azide assists in preventing capping and shedding or internalization of the antibody-antigen complex after the antibodies bind to the receptors). We suggest using our 10X PBS (Code # MB-008 10X PBS pH 7.2 (0.2 M Potassium Phosphate 1.5 M Sodium Chloride)).

  • Fixation Buffer: 2% Paraformaldehyde in PBS.

  • Permeabilization Buffer: 0.1% Triton in PBS.

  • 70% ethanol, stored at -20ºC.

Secondary Conjugated Antibody

  PE
Human Anti-HUMAN IgG [H&L] (GOAT) Antibody Phycoerythrin conjugated
Mouse Anti-MOUSE IgG [H&L] (GOAT) Antibody Phycoerythrin conjugated
Rabbit Anti-RABBIT IgG [H&L] (GOAT) Antibody Phycoerythrin conjugated
Rat Anti-RAT IgG [H&L] (GOAT) Antibody Phycoerythrin conjugated


Isotype Control Antibody

Human FITC
IgA Anti-HUMAN IgA (alpha chain) (GOAT) Antibody Fluorescein Conjugated
IgM Anti-HUMAN IgM (mu chain) (GOAT) Antibody Fluorescein Conjugated
Kappa Anti-HUMAN k (kappa chain) (GOAT) Antibody Fluorescein Conjugated
Lambda Anti-HUMAN λ (lambda chain) (GOAT) Antibody Fluorescein Conjugated


Procedure A: Cell Surface Antigens Analysis

  1. Collect 1-3 x 105 cells per sample, wash with 2 ml FACS buffer.

  2. Centrifuge and aspirate supernatant.

  3. Resuspend cells with 100 µl FACS buffer containing 0.5-1 µg antibody, vortex and incubate on ice for 30min.

  4. Wash cells with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

  5. Resuspend cells with 100 µl FACS buffer containing 1 µg secondary antibody, vortex and incubate on ice for 30min. Perform the incubation in dark. Secondary antibodies used for FACS are typically F(ab?)2 fragment antibodies conjugated to fluorochromes like FITC or R-Phycoerythrin (see our website for a complete listing of suitable reagents).

  6. Wash with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

  7. Resuspend cells with 0.5-2 ml FACS buffer. Place samples in 12 X 75mm Falcon tubes and analyze by flow cytometry as soon as possible (within 1 hr). Alternatively, samples can be fixed with 2% paraformadehyde fixation buffer and stored at 4° C in the dark for up to one week before flow cytometry analysis.

Procedure B: Intracellular Antigens Analysis

  1. Collect 1-3 x 105 cells per sample, wash with PBS.

  2. Fix the cells by adding 100 µl of fixation buffer to cell pellet, vortex and incubate at room temperature for 20 minutes.

  3. Add 1 ml of permeabilization buffer to each tube, centrifuge and aspirate supernatant.

  4. Resuspend cells in 100 µl of permeabilization buffer and incubate at room temperature for 5 minutes.

  5. Wash cells with 2 ml FACS buffer. Centrifuge and aspirate supernatant.

  6. Perform antibody staining as procedure I from step 3 to 7.

If it is desired to simultaneously analyze surface and intracellular molecules at single-cell level, staining cell-surface antigen first by following procedure A step 1 to 6, then stain the intracellular antigen by following procedure B. Perform all procedure B steps in dark.


Procedure C: Staining DNA for Cell Cycle Analysis


This procedure 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.

  1. Collect 1-3 x 105 cells per sample, wash with PBS.

  2. Fix cells in 70% ethanol at 4°C for 30 minutes. For proper fixation and to prevent cell aggregation, employ agitation while fixing.

  3. Centrifuge fixed cells and resuspend pellet in 1 ml of PBS.

  4. Add 100 µl of 200 µg/ml DNase-free, RNaseA and incubate at 37°C for 30 minutes.

  5. Add 100 µl of 1 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 10-30 minutes.

  6. Place samples in 12 X 75mm Falcon tubes and analyze by flow cytometry

Procedure C can be combined with the procedure A of cell surface antigens analysis. Use only FITC-labeled antibodies for surface antigen, since PI emits in the orange to red region of the spectrum (FITC emits green). Do not fix the cells with paraformaldehyde. Start procedure C after step 6 of procedure A.


Additional FACS Protocol Resources

www.protocol-online.org/prot/Cell_Biology/Cytometry