Robosep buffer

PBMC samples were thawed in a 37°C water bath, and cells were immediately and gently added to pre-chilled thaw diluent (1:30 ratio) containing Plasma-Lyte A Injection, pH 7.4 (Baxter Healthcare Corp) with added heparin (10 IU/mL, StemCell Tech) and DNase I (0.01 mg/mL, Stem Cell Tech). Cells were centrifuged at 300x g for 5 min at room temperature and pellet resuspended in 1X RoboSep buffer (StemCell Tech) for subsequent B cell isolation. If cell aggregates were observed, cell suspensions were filtered with a 37 μm cell strainer (StemCell Tech). B cell isolation was performed using the EasySep Human B cell Isolation kit (StemCell Tech) with minor adjustments to the standard protocol, including performing isolation in a 96-well plate with proportionately scaled-down reagents. B cell purity was assessed via flow cytometry for select samples. Cells were stained with: CD19 PE (BD; clone HIB19), CD14 FITC (BD; clone M5E2), CD15 BV510 (BD; clone W6D3), CD3 BV421 (BD; clone SK7), CD56 PE-Cy7 (BD; clone B159), CD45 APC-Cy7 (BD; clone 2D1), and 7-AAD (BioLegend). Samples were acquired on a CytoFlex analyzer (Beckman Coulter), and data were analyzed using FlowJo v10 (FlowJo, LLC).

Single-cell suspensions were obtained from kidney tissues using enzymatic digestion with Liberase (Roche, 05401127001) diluted in Gey’s balanced salt solution. Fascia removal was performed using a 40-µm cell strainer. Next, F4/80⁺ cells were extracted from the cell suspension using the EasySep™ Mouse Biotin Positive Selection Kit II (STEMCELL, 17,665) according to the manufacturer’s instructions. Briefly, the kidney cells were transferred to a 5-mL (12 × 75 mm) polystyrene round-bottom tube and resuspended in RoboSep Buffer (STEMCELL, 20,104), blocked with FcR, and incubated with anti-mouse F4/80 antibody (STEMCELL, 60027BT) for 15 min. Subsequently, the cells were centrifuged, resuspended, incubated with the selection cocktail for 15 min, and reacted with RapidSpheres for 10 min. Finally, the tubes (without lids) were placed into a magnet (STEMCELL, 18,103) for 5 min, the supernatant was discarded, and the cells were collected from the sides of the tube.

The following antibodies from BioLegend were used for flow cytometry: anti-human CD2 (TS1/8), CD3 (HIT3a), CD10 (HI10a), CD33 (WM53), CD34 (581), CD36 (5–271), CD38 (HB-7), CD45 (H130), CD45RA (HI100), CD56 (HCD56), CD94 (DX22), CD122 (TU27), CD132 (TUGh4), CD135 (BV10A4H2), NKG2D (1D11) and NKp46 (9E2). Anti-human NKG2A antibody was from R&D systems (Minneapolis, MN). Anti-mouse CD45 (30-F11) was acquired from BD Biosciences. For flow cytometry, samples were stained with antibodies in 50 μl flow buffer (0.2% bovine serum albumin [BSA, sigma], 0.05% sodium azide [sigma] in PBS) on ice for 30 mins. Stained samples were then analyzed on a BD LSR II flow cytometer, and data analyzed with FACS Diva (BD Biosciences) and FlowJo (TreeStar version e.g. 7.6.5). Isotype-matched control antibodies were used for all fluorochrome-isotype combinations. For fluorescent-activated cell sorting (FACS), cells were stained with appropriate antibodies in RoboSep buffer (Stemcell Technologies), and sorted on a BD FACSAria II (BD Bioscience).

Bone marrow was harvested by flushing the femurs and tibia with RoboSep Buffer (Stemcell Tech, Vancouver, BC, Canada) using a syringe with a 27-gauge needle. Cell clumps were removed by passing the cell suspension through a 70-μm mesh nylon strainer and the cells were centrifuged at 300 × g for 6 min. The pelleted cells were resuspended at 1 × 10⁸ cells/ml, and monocytes were enriched with an EasySep Mouse Monocyte Enrichment kit according to the manufacturer’s instructions.
Isolated monocytes (5 × 10⁵ to 1 × 10⁶) were labeled with PKH26 using a PKH26 Labeling kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. PKH26 fluorescence (a yellow-orange fluorescent dye with long aliphatic tails) technology provides stable incorporation into lipid regions of cell membranes and has been found to be useful for in vitro and in vivo cell tracking applications in a wide variety of systems. In brief, cells were washed once in serum-free RPMI-1640 medium, resuspended in 2 mL kit diluent solution C, mixed with PKH26 at 2 × 10⁻³ mol/L in diluent C, and incubated for 10 min at room temperature in the dark. An equal volume of medium containing 10% FBS was added, and the cells were centrifuged, washed once, and resuspended in serum-containing medium for further analysis.

All experiments using human blood were approved by and carried out in accordance with the Advatta Institutional Review Board. All experimental protocols were approved by the MedImmune Blood Donor Program. Written consent was obtained from healthy adult volunteers who were employees of MedImmune or AstraZeneca, and samples were anonymized for research purposes. Donors positive for HIV infection, hepatitis B or C virus infection, human T-lymphotropic virus infection, or syphilis were excluded from the donor program. Whole blood from healthy volunteers was collected in heparinized CPT tubes. The tubes were spun at 1700 × g for 20 min at room temperature. The supernatant above the plug was collected into new tubes and spun at 300 × g for 20 min with the brake off. The supernatant was discarded, and the PBMC pellet was resuspended in RoboSep buffer (StemCell Technologies, catalog number 20104). The cells were then separated according to the manufacturer’s instructions using negative selection kits for naive CD4 T cells (StemCell Technologies, catalog number 19155), naive CD8 T cells (StemCell Technologies, catalog number 19158), memory CD4 T cells (StemCell Technologies, catalog number 19157), and memory CD8 T cells (StemCell Technologies, catalog number 19159).