Macs running buffer

CD11b⁺ spleen cells were prepared as antigen-presenting cells (APCs). Whole spleen cell suspensions were prepared by passing spleen tissue minced with scissors through a 70-μm cell strainer (BD Biosciences, San Diego, CA, USA). Erythrocytes were lysed in an erythrocyte lysis solution (BD Pharm Lyse™; BD Biosciences) and washed with RPMI-1640 medium containing 10% fetal calf serum (FCS). The cells were then re-suspended in MACS^® running buffer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), incubated with CD11b⁺ magnetic beads (Miltenyi Biotec GmbH) for 15 min at 4°C, and positively sorted using an autoMACS^® Pro Separator (Miltenyi Biotec GmbH). CD11b⁺ cells were re-suspended in RPMI-1640 medium.

For each time-point, cells were detached using TrypLE-express (ThermoFisher, Gibco 12604). Harvested cells were then centrifuged at 300 g and resuspended at the final cell density of 100 cells/mL using a solution containing 40% KnockOut Serum Replacement (KSR, ThermoFisher, Gibco 10828) in DMEM. For each timepoint, two replicates were produced, each containing cells from 4 independent chips that were pooled together then divided in aliquots containing 5000-80,000 cells. Samples were cryopreserved in DMEM supplemented with 40% KSR and 15% DMSO and stored in liquid nitrogen.
scRNA-seq libraries were generated using one or two samples for each replicate. Briefly, each cryopreserved aliquot was thawed at 37 °C until a tiny ice crystal remained in solution. Then each sample was diluted under gentle shaking by dropwise adding 10 volumes of DMEM supplemented with 40% KSR. Cells were washed twice using a washing buffer containing 8% MACS Running Buffer (Miltenyi, 130-091-221) in PBS. Cells were then resuspended in the washing buffer and filtered through a 40 µm cell strainer (Biosigma, 010198Z). Cell viability and concentration were checked by visual inspection using Trypan Blue (Logos Biosystems, L12002).
Single-cell RNA seq libraries were produced according to 10X Single Cell 3’ v2.0 standard protocol and sequenced on Novaseq 6000 (Illumina).

Primary or immortalized preadipoctyes were collected in CellStripper (Corning) buffer, pelleted at 500 × g at 4 °C for 5 min and blocked in MACS Running Buffer (Milteny Biotec) for 10 min on ice. Cells were then incubated with the ASC-1 antibody 10A11 cell culture supernatant at a dilution of 1:2 for 30 min. Cells were incubated with 25 µl protein G microbeads (Milteny Biotec) for 30 min. MS columns (Milteny Biotech) were equilibrated with MACS Running Buffer. The flow through fraction was collected as negative control. The column was washed three times with 200 µl ml MACS Running Buffer, removed from the magnetic field and retained cells were eluted with 500 µl ml MACS Running Buffer. Both flow through and eluted cells were plated on 24-well plates in growth medium and cultured for further experiments.

In order to measure the changes in [Ca²⁺]_i concentration, cells were loaded with Fluo-3AM (Thermo Fisher, Waltham, MA) calcium indicator dye. A Fluo-3AM stock solution of 1.25 mM was prepared in DMSO. Control and treated cells were pelleted and washed in warm MACS running Buffer (Miltenyi Biotec) at a density of 1 × 10⁶ cells/mL. A 4.4 μM working solution of Fluo-3AM was prepared in warm MACS running Buffer and added to the pelleted cells and incubated at 37 °C for 30 min. Cells were washed three times with warm MACS running Buffer and resuspended in 400 μL of 1 mM EGTA solution made with MACS running Buffer to provide Ca²⁺ free cell suspension. The cell suspensions were then transferred to cluster tubes for acquisition on the flow cytometer (BD Biosciences, San Jose, CA).

T cells (CD3+) enrichment was performed using CD3 MicroBeads (catalog no. 130-052-001; Miltenyi Biotec, Auburn, USA) as described previously [21 (link)]. Cell separation with the MACS Separator was carried out according to the manufacturer instructions. Briefly, between 500 μL and 1 mL of cell suspension in RPMI culture medium were used for enrichment. Cells were magnetically-labeled by adding 50 μL MicroBeads per 1 mL of suspension. Cells were incubated at 4–8°C for 15 min and washed subsequently by adding 5 mL of MACS Running Buffer (Miltenyi Biotec) per 1 mL of PB. Cells were centrifuged and resuspended in one volume of buffer. For obtaining higher purity, the whole process was repeated twice. After two purification steps, the positive fraction containing CD3+ cells (85% purity) was used in the mixed lymphocytic reaction.