Histopaque

The PBMC sample was split into two aliquots and CD14 monocytes were isolated from one aliquot and CD19 B cells from the other by magnetic cell sorting using CD14 and CD19 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. CD4 and CD8 T cells were then isolated from the CD14 and CD19 negative fractions, respectively, by magnetic cell sorting using CD4 and CD8 microbeads as described by the manufacturer. The positive selection protocol as outlined takes less than 5 hours from time of blood collection to RNA extraction.
CD16 neutrophils were obtained as follows. Following centrifugation on Histopaque 1077 the red cell/granulocyte pellet was incubated with red cell lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA) on ice for 30 min. Following red cell lysis the granulocytes were recovered by centrifugation, washed with MACS rinsing buffer, and then resuspended in 50 ml MACS running buffer. Neutrophils were then isolated by magnetic cell sorting using CD16 microbeads as described by the manufacturer.

Following euthanasia, mouse legs were removed at the acetabulum and placed into magnesium/calcium-free PBS (Cytiva, #SH30256.02). The bones were cleaned and flushed into a 1.5 mL tube using magnesium/calcium-free PBS. The remaining cells were pelleted via pulse centrifugation then were subject to red blood cell lysis in 250 µL of red blood cell lysis. Following red blood cell lysis, the cells were washed with 1 mL of calcium/magnesium-free PBS then pelleted with pulse spin centrifugation. Cells were then plated for apoptosis at 500,000 cells in 12-well plates in DMEM/F12 media (Thermo Fisher Scientific, 11330032) with an additional 4 mmol/L Glutamax (Thermo Fisher Scientific, 35050061), 5 µg/mL ITS (insulin/transferrin/selenium, SIGMA, I3146), and 8%–9% FBS (VWR, 97068-085). Remaining cells were resuspended in 1 mL of calcium/magnesium-free PBS. For Histopaque gradient, in a 5 mL flow cytometry tube, 1.5 mL of Histopaque 1119 (SIGMA #RNBK6705) was added at room temperature and 1.5 mL of Histopaque 1077 (SIGMA #RNBL3022) at room temperature was carefully pipette on top. Samples were then added on top of the histopaque layers slowly to persevere the interface between the layers. The tubes were then spun at 25°C at 1,000 × g for 25 minutes with no brake. Following centrifugation, the cloudy interface was collected into a 1.5 mL tube and washed with 1 mL of magnesium/calcium-free PBS. The cells were resuspended in 1 mL of magnesium/calcium-free PBS and 150 µL was taken to perform cytopsin. For cytopsin, 150 µL cell suspension was pipetted into funnel and slides were spun at 550 rpms for 1 minute. The areas where cells had adhered were then circled with a wax pen and 150 µL of paraformaldehyde or formalin was added for 15 minutes. The fixing agent was tapped off and then 150 µL of PBS with 0.1% Triton-X was added for 15 minutes. The slides were stained using hematoxylin for 1 minute. The slides then proceed from 70% ethanol for 1 minute, 95% ethanol for 1 minute, 100% ethanol for 3 minutes, xylene for 3 minutes, and finally xylene for 3 minutes. The slides were then allowed to dry and the cover slip was added using cytoseal (Thermo Fisher Scientific #527665). Slides were imaged at 60x and 500 nuclei from each mouse were called for differentiation state in a blinded fashion.