This protocol was used in our Nature Immunology paper.
Method Article
In vitro class switching staining for flow cytometry
https://doi.org/10.1038/nprot.2007.36
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This protocol was used in our Nature Immunology paper.
cytometry
Frosted microscope slides (Fisher Scientific, Pittsburgh, PA)
RPMI-1640 medium (Cellgro)
ACK Lysing Buffer (Cambrex)
Cell strainers, 70 μm nylon (Becton Dickinson)
CD43 (Ly-48) magnetic beads and separation columns (Miltenyi)
B cell complete medium:
RPMI-1640 medium (Cellgro) plus 2 mM glutamine (Gibco) and 100 U/ml of penicillin g/ml and 100 μg/ml of of streptomycin (Gibco), 10% fetal calf serum (heat inactivated; Sigma), and 5×10-5 M 2-mercaptoethanol (Sigma).
24-well flat bottom culture plates (Nunc)
Anti-CD40 (clone 3/23; Pharmingen)
Recombinant mouse IL-4 (Peprotech)
Fc blocker (anti-CD16/CD32; Pharmingen)
Biotinylated anti-IgG1, anti-IgG2a-2b (both Pharmingen)
Streptavidin conjugate (Pharmingen)
FACS buffer: PBS + 0.5% fetal calf serum + 5mM EDTA
Perform the following steps at 4 oC unless indicated otherwise.
1 Harvest splenocytes by grinding of spleens in RPMI-1640 medium between two frosted microscope slides. Spin cells at 1000 RPM for 5 min. Remove erythrocytes by adding one equivalent volume of ACK Lysing Buffer to the remaining pellet. Gently resuspend, swirl tube for 1 min. Fill tube with 10 ml of RPMI-1640, centrifuge at 1000 RPM for 5 min. Resuspend in 10 ml of RPMI-1640. Filter through 70 μm nylon cell strainer. Centrifuge at 1000 RPM for 5 min. Resuspend 1 × 107 cells in 500 μl of FACS buffer. Obtain B cells by CD43 negative purification using anti-CD43 magnetic beads.
2 Dilute CD43- B cells to a concentration of 5×105 cells/ml in B cell complete medium. Use 24-well plates. Incubate 5×105 B cells in presence of 10 μg/ml of anti-CD40 (clone 3/23), plus 10 ng/ml of recombinant mouse IL-4 for IgG1 analysis (6). Incubate for 4 d in a humidified 37 oC, 5% CO2 incubator.
3 Wash cells with FACS buffer. Preincubate cells on ice for 10 min in presence of Fc blocker (anti-CD16/CD32). We usually use 0.5 μl of Fc blocker/ 5×105 cells in a total volume of 100 μl. CRITICAL STEP: Good Fc blockade.
4 Stain cells with biotinylated anti-IgG1, anti-IgG2a-2b (we used the antibodies at a dilution of 1:200 in a total volume of 100 μl). Incubate on ice for 20 min. Wash 2x with FACS buffer.
5 Stain cells with streptavidin conjugate of your choosing. Wash 2x with FACS buffer.
6 Analyze by flow cytometry.
Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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