Anti pe microbeads ultrapure

Peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and healthy donor buffy coats were isolated by density gradient centrifugation using Cytiva Ficoll-Paque PLUS Media (Thermo Fisher). Isolated PBMCs from COVID-19 patients were frozen with CTL kit (cat: CTLC-ABC, ImmunoSpot) and then stored in liquid nitrogen until use. T cells were isolated from PBMCs of healthy donor buffy coats by using the Pan T Cell Isolation Kit (Miltenyi Biotec). Vα7.2⁺ cells from PBMCs of healthy donor buffy coats were enriched via positive selection with Anti-PE MicroBeads UltraPure (Miltenyi Biotec) after staining with PE-labeled Vα7.2 monoclonal antibody (clone: 3C10, Biolegend).
The purity of T cells and classical MAIT-cell contents of the Vα7.2⁺ enriched cells used in the in vitro viral challenges was assessed by CD3 or 5-OP-RU loaded MR1 tetramer staining (NIH Tetramer Core Facility, Emory University, Atlanta, GA, The USA) (Corbett et al., 2014 (link)) and flow cytometry analysis. 31 % of the Vα7.2⁺ enriched cells were MR1-tetramer positive classical MAITs (Supplementary Fig. 1A) and > 90 % of the magnetic bead isolated T cells were CD3-positive (Supplementary Fig. 1B).

To isolate pulmonary mouse MAIT cells, non-parenchymal lung mononuclear cells (LMNCs) from indicated B6-MAIT^CAST cohorts were subjected to magnetic cell sorting. Briefly, phycoerythrin (PE)-conjugated, 5-OP-RU-loaded mouse MR1 tetramers were used to stain MAIT cells [7 (link),42 (link)], which were then purified using Anti-PE MicroBeads UltraPure, LS Columns and a QuadroMACS Separator (Miltenyi Biotec). Isolated MAIT cells were always 90–99% pure after two separation rounds (S1 Fig). Cells were washed in PBS containing 10% BSA and 50 mM ethylenediaminetetraacetic acid before pellets were flash-frozen and stored at -80°C.
Total RNA was extracted using a PicoPure RNA Isolation Kit (Thermo Scientific) and converted to cDNA using SuperScript IV VILO Master Mix with ezDNase (Thermo Scientific). cDNA and TaqMan Fast Advanced Master Mix were added to each well of a custom-made, 96-well TaqMan Array Fast Plate (Thermo Scientific) containing lyophilized primer/probe sets listed in S1 Table. cDNA was amplified per manufacturer’s instructions, and cycle threshold (Ct) values were generated using a StepOne Plus Real-Time PCR System (Applied Biosystems). Normalized ΔCt values were determined by subtracting each Ct value by that of Gapdh, and the following formula was employed to calculate the relative mRNA content of MAIT cells for indicated genes: Fold Change = 2^-(ΔΔCt).

PDGFRA negative selection was performed using Anti-PE MicroBeads UltraPure (130-105-639; Miltenyi Biotec), LS Columns (130-042-041; Miltenyi Biotec) and a MidiMACS Separator (130-042-302; Miltenyi Biotec). Then, ITGA8 positive selection was performed using Anti-Biotin MicroBeads UltraPure (130-105-637; Miltenyi Biotec), MS Columns (130-0042-201; Miltenyi Biotec) and MiniMACS Separator (130-042-102; Miltenyi Biotec). To assess the differentiation ability of the sorted cells, 3 × 10⁵ ITGA8−/PDGFRA- and ITGA8+/PDGFRA- cells were aggregated in differentiation medium with 1 µM CHIR and 5 ng/mL human FGF9 overnight at 37 °C and then co-cultured with mouse embryonic spinal cords for nine days.

Cardiac γδT cells were enriched from total cardiac cells using anti-PE Microbeads UltraPure (Miltenyi Biotec) after staining with PE-conjugated anti-mouse γδTCR (BD). Isolated cardiac γδT cells were transferred to 96-well Maxisorp plate (Nunc), which was coated overnight with 100 μl of 4-μg/ml anti-CD3 antibody (145-2C11; TONBO Biosciences) and cultured in the presence of 10-ng/ml mIL-1β (Peprotech) and 10-ng/ml mIL-23 (RD systems). After 48 h, the supernatants were collected and assayed for IL-17A production using LEGEND MAX^TM ELISA Kit (BioLegend) according to the manufacturer’s instructions.

After isolation of mononuclear cells, CD4⁺ T cells were purified using PE anti-CD4, followed by anti-PE ultrapure microbeads (Miltenyi Biotec), and sorted using the AutoMACS Pro sorter (Miltenyi Biotec). Splenic DCs were purified by using PE anti-CD11c, followed by anti-PE ultrapure microbeads, and sorted as described above. CD4⁺ T cells were co-cultured with splenic DCs (CD4⁺ T cells:splenic DCs = 4:1) in the presence of 100 µg/mL β-galactosidase (Sigma-Aldrich, St. Louis, MO, USA) for 4 days. IL-17A and IL-22 in the supernatant were detected by enzyme-linked immunosorbent assay kits (R&D systems) according to the manufacturer’s instructions.

Single-cell suspensions from BM (femora and tibiae of both hind legs) and spleen were prepared. T cells were depleted from BM (T cell depleted bone marrow; TCDBM) using anti-CD90.2 MicroBeads (Miltenyi Biotec). CD4⁺CD25⁻ conventional T cells (Tconv) were isolated from splenocytes by depletion of CD25⁺ cells using anti-CD25-PE (antibodies are listed in Supplementary Table 2) and anti-PE UltraPure MicroBeads (Miltenyi Biotec), followed by enrichment of CD4⁺ cells from the CD25⁻ fraction through labeling with anti-CD4 MicroBeads (Miltenyi Biotec). For short-term (d7) FACS and RNA-seq experiments BALB/c recipients were irradiated (8 Gy) on the day of BMT and transplanted i.v. with 2.5 × 10⁶ TCDBM and 1 × 10⁶ Tconv from B6-CD45.1 mice with or without in vitro expanded allo or polyTreg from Foxp3^gfp mice at a 1:1 ratio. Recipients of the “no GvHD” group received TCDBM and Treg only. In long-term survival experiments irradiated BALB/c recipients were transplanted with 2.5 × 10⁶ TCDBM on d0 and 0.25 × 10⁶ Tconv on d2 for GvHD induction. Mice of the GvHD prophylaxis groups (allo or poly) received additional 0.25 × 10⁶ in vitro expanded Treg from B6 WT mice on d0. Recipients were monitored daily, body weight and GvHD symptoms assessed twice weekly by non-blinded investigators applying a standardized scoring system^{63 (link)}.

For 10x Genomics single-cell gene expression and immune profiling, we used PBMCs isolated from 60 ml of blood of donor M1 before the second immunization. PBMCs were stained with NS4B-dextramer-PE (Immudex) according to the manufacturer’s protocol. Additionally, cells were stained with anti-CD3-eFluor450 (eBioscience), and anti-CD8-FITC (eBioscience). Previous to FACS sorting procedure, we used propidium-iodide to mark dead cells. As the NS4B-specific cell frequency was very low (Figure 4—figure supplement 1C), we used anti-PE Ultra-pure MicroBeads (Miltenyi) for the enrichment. In brief, every milliard of PBMCs was incubated with 10 µl of magnetic beads for 15 min on ice. After a washing step with PBS 5% FCS, the cell suspension was applied on MS MACS Column (Miltenyi). Columns were washed three times with PBS 5% FCS and stained with propidium-iodide just before the FACS (FACS Aria II). This procedure resulted in a dramatic increase of NS4B-specific cell frequency in the sample (Figure 4—figure supplement 1C) and accordingly lead to reduced FACS procedure time. For single-cell immune profiling of bulk T-cell clonotypes from PBMCs, we stained the cells with anti-CD3-eFluor450 (Invitrogen) and propidium-iodide, thus selecting CD3 positive cells. Approximately 10,000 CD3+ cells were used for 10x Genomics VDJ T-cell receptor enrichment protocol.