Apc conjugated anti gr1

The neutrophils from the vaccinated animals were evaluated 30 days after the challenge with M. tuberculosis. The splenocytes were obtained as described above. The cells were incubated for 30 min with APC-conjugated anti-GR1 (BD PharMingen), PercP-conjugated anti-CD14, PE-conjugated anti-CD11c and FITC-conjugated anti-CD11b antibodies (eBioscience). Then, the cells were washed with PBS and fixed with paraformaldehyde using Perm Fix (BD Cytofix). A total of 100,000 events were acquired using BD Biosciences FACSVerse^TM and analyzed with the FlowJo 9.0 Software. The neutrophils percentage was determined evaluating singlets that were GR-1⁺, CD11c^-, and CD14^-. The total number of neutrophils was obtained multiplying the percentage of neutrophils by the total number of splenocytes from each animal.

WBM was labeled with PB-conjugated anti-B220, PE-conjugated anti-CD4, PE-conjugated anti-CD8, BUV395 conjugated anti-TER 119, APC-conjugated anti-GR1 (BD Biosciences), and Alexa Fluor 488 conjugated anti-CD11b (BioLegend) antibodies. For the B220 double sort discard population, B220 positive (B220 +) cells were isolated by FACS. This primary sorted B220 + population was centrifuged at 1300 rpm for 10 min at 4 °C, the pellet re-suspended in PBS and subjected to reanalysis using the same gating schema as the primary sort. Two populations of cells on the re-analysis, those persistently positive for the B220 and those negative for the B220 (the B220 double sort discard population), were analyzed for the presence or absence of the remaining Lin + markers by flow cytometry. GR1 + cells isolated on primary sort were similarly subjected to double sorting and Lin + marker analysis as above. For stem cell marker analysis, WBM was incubated with APC-conjugated antibodies against either GR1 or B220 (BD Biosciences), and FITC-labeled anti-c-Kit (BD Biosciences), PB-labeled anti- Sca-1 (BioLegend) and PE-labeled anti-CD150 (BioLegend) antibodies. Primary sorted GR1 + cells or B220 + cells were then re-analyzed and percent stem cell marker positive cells in the different cellular populations was determined by flow cytometry.