SRBC Protocols

Detection of SRBC-specific antibodies in serum

  1. Prepare SRBC
    • Mix SRBC gently and well (not too hard)
    • Wash 1ml of blood with 50ml PBS 2 times: 2000rpm/10min (Prog.8) -> for 400 uL packed blood
    • Add 3600 uL PBS to 400 uL packed blood
  2. Pipette 20µl SRBC from the final 4ml for each sample into mini FACS tubes
  3. Add sera
    • Add 4ul serum into 36ul PBS and take 10, 4, 2, and 1 ul, so there will be 4 dilutions per sample
    • Controls: (1) SRBC without serum, stained with antibody, (2) SRBC with serum, without antibody
  4. Incubate on ice for 20 minutes
  5. Wash 1x
  6. Add anti-IgM APC and anti-IgG1 PE at 1:100
    • Optional: anti-B220 APC780 as a negative control
  7. Incubate on ice, in the dark for 20 minutes
  8. Wash with FACS buffer 1x
  9. Resuspend in 600-800 uL FACS buffer
  10. Measure on flow cytometer
  11. For analysis, plot MFI of IgM and IgG1 against serum dilution and choose the dilution that is within the linear range (can compare several wild type mice to determine dilution to use)
    • Compare MFI of all samples at the same dilution to see IgM and IgG1 SRBC-specific antibody production

Detection of SRBC-binding B cells

  1. Prepare SRBC
    • Mix SRBC gently and well (not too hard)
    • Wash 1ml of blood with 50ml PBS 2 times: 2000rpm/10min (Prog.8) -> for 400 uL packed blood
    • Add 3600 uL PBS to 400 uL packed blood
  2. Incubate 50 ul SRBC with 1 ml of 10 uM eFluor670 in PBS at room temperature for 10 minutes
  3. Wash SRBC loaded with eFluor670 with FACS buffer and resuspend in 1 ml FACS buffer for staining
  4. Wash splenocytes and incubate in 100 µl FACS buffer with primary antibodies for 20 minutes on ice
    • Primary antibodies used: 1:100 anti-B220 eFluor780, 1:100 anti-FAS PE-Cy7, and 1:100 GL7-Biotin
  5. Wash and resuspend splenocytes in 100 µl FACS buffer with 1:100 Streptavidin-PE and 20 ul SRBC loaded with eFluor670
  6. Stain splenocytes for 20 minutes on ice, then wash and resuspend in FACS buffer
  7. Measure on flow cytometer
    • Doublet discrimination was not performed during analysis.