Recommended Assay Procedure:
Immunofluorescent Staining of Mouse Intracytoplasmic/Intracellular IgM:
1. Prepare a single-cell suspension and determine cell number.
2. Suspend cells at 2×10e7 cells/ml in BD Pharmingen™ Stain Buffer (Cat. No. 554656) [or alternatively, prepare a staining buffer made up in PBS + 2% FBS + 0.1% sodium azide] and transfer to U-bottom microwell plates in 50 µl/well for immunofluorescent staining.
3. Block Fcγ receptors by adding 0.2 µg of BD Fc Block™ (Cat. no. 553141) in 50 µl of staining buffer to each well.
4. Incubate 5 minutes on ice.
5. Add 200 µl of staining buffer/well and resuspend cells. Centrifuge at 250×g for 5 minutes and aspirate supernatant.
6. Block cell surface IgM with purified rat anti-mouse IgM (clone II/41) (Cat. No. 553435) by adding 1.0 µg per sample in 50 µl of staining buffer/well.
Note: Surface markers may be stained during this step as described in the "Immunofluorescent Staining of Mouse and Rat Leukocytes for Flow Cytometry" in the Technical Protocols section of our web site at http://www.bdbiosciences.com/pharmingen/protocols/Mouse_and_Rat_Leukocytes.shtml
7. Incubate 15 minutes on ice.
8. Wash 2× as described in Step 5.
9. Resuspend cells in 100 µl of BD Cytofix/Cytoperm™ solution (Cat. No. 554714) per well.
10. Incubate 30 minutes at room temperature.
11. Wash 2× with 200 µl of 1× BD Perm/Wash™ buffer (Cat.No. 554714) per well. Centrifuge at 250×g for 5 minutes and aspirate supernatant between washes.
12. Stain for intracellular IgM by adding ≤ 1 µg of FITC rat anti-mouse IgM (clone II/41) antibody in 50 µl of 1× BD Perm/Wash™ buffer/well.
Note: Other antibodies recommended for staining of intracellular markers may be added during this step as described in Step 12.
13. Incubate for 30 minutes at room temperature.
14. Wash 2× as described in Step 11.
15. Resuspend and transfer samples in 100 µl of staining buffer to tubes appropriate for analysis with a flow cytometer. Bring volume in each tube to 400 µl with staining buffer.
16. Analyze samples on a flow cytometer.
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