Live dead fixable near ir cell stain kit

Macrophages were pre-incubated with LIVE/DEAD Fixable Near-IR cell stain kit (Invitrogen, L34976) and with Mouse BD Fc Block™ (≤ 1 μg/million cells in 100 μL, BD Biosciences #553141) at 4°C for 5 min and then stained with Pacific Blue anti-CD45 (5 µg/mL, BioLegend #109820), anti-CD11b FITC (5 µg/mL, BD Biosciences #557396), anti-CD11b APC-cy7 (2 µg/mL, BioLegend #101226), anti-F4/80 APC (5 µg/mL, BioLegend 123116), or anti-F4/80 PE (2 µg/mL, BD Pharmigen 565410) antibodies. After cells were fixed with formaldehyde 2% and permeabilized with permeabilization buffer (eBioscience, #00-8333-56), ingested erythrocytes were stained intracellularly with anti-TER-119 PE (2 µg/mL, Stemcell #60033) antibody. Stained cells were analyzed using the LSRFortessa (BD) or the SP6800 Spectral Analyzer (Sony). Data were analyzed using FlowJo and FCS express 6 (De Novo software).

Cells were preincubated with a LIVE/DEAD Fixable Near-IR cell stain kit (Invitrogen) and with Mouse BD Fc Block™ (≤1 μg/million cells in 100 μl, BD Biosciences) at 4 °C for 10 min.
The following antibodies were purchased from BD Bioscience: anti-CD45 (clone 30-F11), anti-CD19 (clone 1D3), anti-CD3 (clone 17A2), anti-CD25 (clone 7D4), anti-CD11c (clone HL3), and anti-I-A/I-E (clone M5/114.15.2). The following antibodies were purchased from BioLegend: anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), anti-CD11b (clone M1/70), anti-CD115 (AFS98), anti-CD11c (clone N418), F4/80 (clone BM8), anti-Ly-6G (clone 1A8), and anti-CD69 (clone H1.2F3). The following antibody was purchased from eBioscience: anti-CD4 (clone GK1.5). Corresponding isotype-matched irrelevant specificity controls were purchased from BD, BioLegend, and eBioscience. Reticulocytes were stained with thiazole-orange (Sigma-aldrich). Multiparameter analysis was performed with an LSRFortessa analyzer (BD Biosciences), a SP6800 Spectral Analyzer (Sony) or an Aurora 5 L spectral flow cytometer (Cytek). The data were analyzed using FlowJo software (version 10.7.1) and FCS express 7 (De Novo software).