If there are few cell events, see troubleshooting: problem 4

If there are few cell events, see troubleshooting: problem 4. Open in a separate window Figure?2 Flow cytometry capture of gp140-specific B cell populations (A) Gating of CD19+ IgD- B cells to identify background levels of PE positivity from a sample lacking PE probe staining. (B and C) Representative gating of sequentially stained PBMC with biotinylated gp140 followed by SA-PE. is a general calculation for gp140 proteins. If desired, an exact calculation can be done on a per gp140 basis. Using the smallest volume possible, add enough SA-PE to your gp140 of interest to make a 4:1 molar ratio of biotinylated gp140:SA-PE. This will yield a tetramer with 4 gp140 epitopes bound to an GNE-493 SA-PE (Morris et?al., 2011). Can use streptavidin-allophycocyanin (SA-APC) instead of SA-PE, or can combine SA-PE and SA-APC (4?L of each) with 492?L FACS wash buffer to select double positive events to reduce background. Additional positive control tubes using other mAbs (PGT128, 4E10, etc.) can be included to test the probes specific reactivity to antibodies that recognize various epitopes. Alternatively, Ramos cells stably transfected to express VRC01 IgM (positive control) or irrelevant antibodies such as anti-influenza or anti-coronavirus antibodies or no antibody (negative control) can be used in place of beads (Benjamin et?al., 1982; Lingwood et?al., 2012). for 5?min at room temperature (20CC22C). 4. Using a P1000 pipettor, aspirate the top 3?mL of R10 media from the FACS tubes. 5. Vigorously vortex the FACS tubes. 6. Centrifuge the stock of Purified Mouse Anti-human IgG for 1?min at 1,000? for 5?min at room temperature. 9. Using a P1000 pipettor, aspirate all but 50C100?L R10 media. 10. Repeat steps 8 and 9. 11. Vigorously vortex then centrifuge the stock of VRC01 for 1?min at 1,000? If available, add 1?g of an irrelevant human mAb to the negative control tube. for 5?min at room temperature. 14. Using a P1000 pipettor, aspirate all but 50C100?L R10 media. 15. Repeat steps 13 and 14. 16. Add 5?L of tetramer probe mix to all tubes. Incubate for 15?min at 4C in the dark. 17. Fill FACS tube with 4?mL R10 media. Vigorously vortex and centrifuge at 300? for 5?min at room temperature. 18. Using a P1000 pipettor, aspirate all but 50C100?L R10 media. 19. Repeat steps 17 GNE-493 and 18 four more times. 20. Store the test and negative control tubes in the dark at 4C until the beads can be tested on a flow cytometer. 21. Compare the negative control to the positive control. If the positive control MFI is distinctly above the negative control MFI (Figure?1), these probes can be used to isolate probe-specific B cells as previously described (Morris et?al., 2011). If the probes do not bind to the positive control beads as a distinct population from the negative control (see troubleshooting; problem 1), epitopes may be occluded from BCR binding. Therefore, CR6 test the ability of the antigen to be used GNE-493 in the sequential staining method below. If there are high levels of background, positive samples are negligibly positive, or there are few bead events see troubleshooting: problem 2, problem 3, or problem 4, respectively. Open in a separate window Figure?1 Quality control bead assay using pre-conjugated tetramer staining or sequential staining (Top) Example of failed gp140 tetramer binding and (bottom) successful GNE-493 use of sequential staining of gp140 in a bead assay. Components for bead assays are listed in the black box. Anti-mouse beads are used to capture mouse anti-human IgG. VRC01 is then captured by the mouse anti-human antibody. Beads are then used to test pre-conjugated gp140 tetramer probes (top panel). If the probes do not bind to VRC01 bound beads (red histogram) as a distinct population from unstained negative control beads (blue histogram), sequential staining should be performed using biotinylated gp140 incubated with VRC01 conjugated beads followed by incubation with SA-PE (bottom panel). We recommend collecting 20,000 events for all samples tested to ensure an accurate result. Additional positive control tubes using other mAbs (PGT128, 4E10, etc.) can be included to test the probes specific reactivity to antibodies that recognize various epitopes. Alternatively, Ramos cells stably transfected to express VRC01 IgM (positive control) or irrelevant antibodies/no antibody (negative control) can be used in place of.