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Flow Cytometry Protocol for Staining Membrane-associated Proteins in Suspended Cells
Phenotyping suspended cells based on antigens present on the membrane is perhaps the most common use for flow cytometry. Because membrane proteins are readily accessible to the antibody, permeabilization steps are not required. However, experimental conditions, such as antibody concentration, incubation time, and temperature, should be optimized for each flow cytometry experiment. The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for the staining of membrane-associated proteins.
Please read the protocol in its entirety before starting.
Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions)
Flow Cytometry Red Blood Cell Lysis Solution
Flow Cytometry Human Lyse Buffer (10X; Catalog # FC002) or Flow Cytometry Mouse Lyse Buffer (10X; Catalog # FC003) or equivalent
Flow Cytometry Staining Buffer
R&D Systems, Catalog # FC001, or an equivalent solution containing BSA and sodium azide
Fluorochrome-conjugated antibodies suitable for use in flow cytometry
Isotype Control Antibodies
FACS™ Tubes (5 mL round-bottom polystyrene tubes)
Pipette Tips and Pipettes
Sample preparation: For staining peripheral blood cells, whole blood should be collected in evacuated tubes containing EDTA or heparin as the anticoagulant. Contaminating serum components should be removed by washing the cells three times in an isotonic phosphate buffer (supplemented with 0.5% BSA) by centrifugation at 500 x g for 5 minutes. For staining cell lines or cells from activated cell cultures, the cells should be centrifuged at 500 x g for 5 minutes and washed three times in an isotonic PBS buffer (supplemented with 0.5% BSA) to remove any residual growth factors that may be present in the culture medium. Adherent cell lines may require pretreatment with 0.5 mM EDTA to facilitate removal from their substrates. Cells that require trypsinization to enable removal from their substrates should be further incubated in medium for 6-10 hours on a rocker platform to enable regeneration of the receptors. The use of the rocker platform will prevent reattachment to the substrate.
Note: Titration experiments should be performed to determine optimal reagent amounts.
Harvest cells and aliquot up to 1 x 106 cells/100 μL into FACS tubes. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 15 minutes at room temperature.
Note: Do not wash excess blocking IgG from this reaction.
Add conjugated antibody (10 μL/106 cells, or a previously titrated amount) and vortex. Incubate cells for 30 minutes at room temperature in the dark.
Remove any unbound antibody by washing the cells in Flow Cytometry Staining Buffer. Centrifuge the suspended cells at 300 x g for 5 minutes and decant the buffer. Resuspend the cells by adding 2 mL of Flow Cytometry Staining Buffer. Repeat the wash two times.
Note: If using whole blood, samples should go through a red blood cell lysis step at this point using Flow Cytometry Human or Mouse Lysis Buffer.
Lysis of Red Blood Cells: Add 2 mL of 1X Human (Catalog # FC002) or Mouse (Catalog # FC003) Lyse Buffer to each tube, vortex, and incubate in the dark at room temperature for 10 minutes. Centrifuge and wash cells in Flow Cytometry Staining Buffer as described in step 4 above.
Note: If an unconjugated primary antibody was used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in Flow Cytometry Staining Buffer, starting with the suggested concentration in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 4.
Resuspend the cells in 200 – 400 μL of Flow Cytometry Staining Buffer for final flow cytometric analysis.
Note: For a negative control, a separate set of cells should be stained with an isotype control antibody using the steps outlined above.
B220/CD45R Expression in Mouse Splenocytes. Mouse splenocytes were stained with an APC-conjugated Rat Anti-Mouse B220/CD45R Monoclonal Antibody (Catalog # FAB1217A; filled histogram) or an APC-conjugated Rat IgG2A Isotype Control (Catalog # IC006A; open histogram). B220 antigens represent a subset of mouse CD45 isoforms predominantly expressed on all B lymphocytes, including pro-, mature, and activated B cells. The B220/CD45 antigen antibody is commonly used as a B cell marker.
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