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Home » Resources » Protocols » Flow Cytometry Protocol for Staining Membrane-associated Proteins in Suspended Cells

Flow Cytometry Protocol for Staining Membrane-associated Proteins in

Suspended Cells
Phenotyping suspended cells based on antigens present on the Literature
membrane is perhaps the most common use for flow cytometry. Product Categories
Because membrane proteins are readily accessible to the
antibody, permeabilization steps are not required. However, Protocols
experimental conditions, such as antibody concentration,
incubation time, and temperature, should be optimized for each Chromatin
Immunoprecipitation (ChIP)
flow cytometry experiment. The following protocol has been
Protocol
developed and optimized by R&D Systems Flow Cytometry
Laboratory for the staining of membrane-associated proteins. Flow Cytometry
Protocols
Please read the protocol in its entirety before starting.
Protocol For Analysis of
Cell Viability using 7-
Amino Actinomycin D (7-
AAD)

Protocol For Analysis of

Cell Viability using
Reagents Required Propidium Iodide

Fc Receptor Blocking Reagents (These include Fc receptor blocking antibodies or IgG solutions) Protocol For Staining
Flow Cytometry Red Blood Cell Lysis Solution Intracellular Molecules
using Alcohol to
Permeabilize the Cell
Flow Cytometry Human Lyse Buffer (10X; Catalog # FC002) or Flow Cytometry Mouse Lyse Buffer Membrane
(10X; Catalog # FC003) or equivalent
Protocol For Staining
Flow Cytometry Staining Buffer Intracellular Molecules
using Detergents to
Permeabilize the Cell
R&D Systems, Catalog # FC001, or an equivalent solution containing BSA and sodium azide
Membrane

Fluorochrome-conjugated antibodies suitable for use in flow cytometry Protocol For Staining
Isotype Control Antibodies Membrane-associated
Proteins in Suspended
Cells
Materials Required
IHC/ICC Protocols
FACS™ Tubes (5 mL round-bottom polystyrene tubes)
Pipette Tips and Pipettes Proteome Profiler Arrays
Centrifuge
Vortex

Procedure

1. 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.

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 Note: Titration experiments should be performed to determine optimal reagent amounts.

2. 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.

3. 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.
4. 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.

5. 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.

SAMPLE DATA

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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FACS is a trademark of Becton Dickinson and Company

Flow Cytometry
Related Information

Flow Cytometry Protocols

Flow Cytometry Supplemental reagents

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