Immunostaining and flow cytometry: FITC Rat Anti-Mouse IgG2b may be used as a primary or secondary reagent in immunofluorescent staining. For detection of intracytoplasmic IgG2b, please refer to the following protocol.
Immunofluorescent Staining of Intracellular Immunoglobulin (Ig) Protocol
1. Prepare a single-cell suspension and determine cell number.
2. Suspend cells in staining buffer (PBS + 2% FBS + 0.1% Sodium Azide, Stain buffer, Cat. No. 554656) at 2 × 10e7 cells/ml and transfer to U-bottom microwell plates in 50 µl/well for immunofluorescent staining.
3. Block Fcγ receptors by adding 0.2 µg of Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™), (Cat. No. 553141/553142) 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 surface Ig with Purified Rat Anti-Mouse IgG2b (Cat. No. 553392) 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 "Immunofluorescent Staining of Mouse and Rat Leukocytes for Flow Cytometry" under the protocols section of "Multicolor Flow Cytometry" at our website: http://www.bdbiosciences.com/us/s/resources.
7. Incubate 15 minutes on ice.
8. Wash 2X as described in Step 5.
9. Resuspend cells in 100 µl of BD Cytofix/Cytoperm™ intracellular staining buffer (BD Cytofix/Cytoperm™ Kit, Cat. No. 554714) per well.
10. Incubate 30 minutes at room temperature.
11. Wash 2X with 200 µl of 1X Perm/Wash buffer (provided in the BD Cytofix/Cytoperm Kit) per well. Centrifuge at 250 x g for 5 minutes and aspirate supernatant between washes.
12. Stain intracellular Ig by adding 1 µg of FITC Rat Anti-Mouse IgG2b in 50 µl of 1X 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 2X 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.