Helix np blue

Spleens were extracted and stored in cooled FACS Buffer (PBS, 10 mM EDTA, 5% FBS) until splenocytes isolation. The spleens were dissected, squashed against 70 μm cell strainer and washed with cold FACS Buffer. Cells were then centrifuged twice (3 min, 500g, 4°C). The pellet was re-suspended with red blood lysis buffer (Sigma-Aldrich, Israel), incubated for 3 min at room temperature, centrifuged (3 min, 500g) and re-suspended with FACS buffer containing 50 μg/ml gentamycin to eliminate extracellular bacteria or with Brilliant stain buffer (BD biosciences) (several brilliant violet fluorophores were introduced in one panel). Single cell suspensions were incubated with CD16/CD32 blocking antibodies (Biolegend, Israel) for 15 minutes on ice. Subsequently fluorophore conjugated antibodies cocktails were introduced and incubated for 30 minutes on ice. Cells were washed, re-suspended with 500-1000 mL FACS buffer and passed through a cell strainer. Prior to analysis, live/dead stain was added to the samples (final concentration of 30 nM, Helix NP Blue, BioLegend, Israel). For absolute quantification, 10-50 μL of BD Liquid Counting Beads (BD Biosciences) were added to the sample prior analysis to a fixed final concentration.

Cell acquisition was done on either BD FACSAria III (BD Biosciences) or by Cytec Aurora (Cytek Biosciences). Cell sorting was performed using a BD FACSAria III (BD Biosciences), using gating of live/dead (Helix NP Blue, Biolegend) and doublet cells. Single cells were sorted into 384 wells plate containing 2 μL of solution containing barcoded poly T reverse transcription primers as described (Jaitin et al., 2014 (link)). For bulk cell capture, 1000 cells from each population was sorted into tubes containing 50 μL lysis buffer (0.1% Triton-100, 0.5U/μl RNasin+ (promega). Immediately after sorting, plates were spun down, flash frozen in a mixture of dry ice and ethanol and stored in −80°C until processing. Flow cytometry data was analyzed using the FlowJo Software.