Pe anti mouse cd19 antibody

T cell population detection post vaccination was assessed using flow cytometry assay and 50 μL whole EDTA blood. Red blood cells were lysed with ACK buffer and washed twice with 1% (v/v) FBS in 1× PBS. The fluorophore-conjugated antibody mixture was added to the sample and incubated on ice for 20 min. FITC anti-mouse CD3ε Antibody (Cat#10036), APC/Cyanine7 anti-mouse CD8a Antibody (Cat#100714), APC anti-mouse CD4 Antibody (Cat#100412), and PE anti-mouse CD19 Antibody (Cat#115508) were bought from Biolegend (San Diego, CA, USA). Cells were then resuspended with 1% FBS in 1× PBS after washing 3 times. All samples were run on the flow cytometer.

Wild-type and MIDN KO HEK-293T cells were infected with lentivirus encoding the pInducer20 system expressing mouse CD19 as a doxycycline-inducible marker for cell-surface staining. After infection, the cells were treated with doxycycline (100 ng/mL) for 2 days and were stained on the cell surface using PE anti-mouse CD19 antibody (Biolegend, 152407, RRID: AB_2629816) for 30 minutes using 1 μL of antibody diluted in 100 μL of media containing 1 million cells. The cells were rinsed twice with PBS before sorting for the PE-positive population. The sorted cells were allowed to expand for more than 1 week before restaining the cells using PE anti-mouse CD19 antibody in the absence of doxycycline induction. The PE-negative population was sorted, and the cells were expanded before another round of staining and sorting for the PE-negative population. This allowed for a population of cells that responded to doxycycline with minimal leakiness. Finally, lentivirus encoding the pHAGE TRE-Midnolin was infected into these MIDN KO HEK-293T cells and selected with blasticidin to allow for stable, doxycycline-inducible expression of midnolin.