Pe cy7 rat anti mouse cd8a

PDA tumor growth in KPC mice was monitored every month using small-animal ultrasound (Vevo770, VisualSonics, Toronto, Ontario, Canada). The tumor was harvested when reaching 10 mm in size, minced, and dissociated in pre-warmed digest medium [5% FBS RPMI 1640 with collagenase (1500 U/ml), and hyaluronidase (1000 U/ml); Life Technology, Carlsbad, CA, USA] and incubated at 37 °C for 1 h. Tumor cells after digestion were filtered through a cell strainer, centrifuged, and washed with cold PBS.
TAMs were sorted by the Flow Cytometer. The cell suspension was stained with a mixture of PE-conjugated anti-mouse F4/80 antibody (Biolegend), APC-conjugated anti-mouse CD3 antibody (Biolegend), PE-Cy™7 rat anti-mouse CD8a (BD Biosciences, San Jose, CA, USA), FITC-conjugated anti-CD4 antibody (Biolegend), and PI (BD Biosciences) for 30 min. CD3-F4/80⁺ live cells were selected for analysis.

The following reagents were used in this study: wild-type smooth strain Escherichia coli (E. coli) (O55:B5, ATCC® 12014™), LPS from E. coli (0111:B4) and RIPA buffer with protease inhibitor cocktail were from Sigma-Aldrich, St Louis, Missouri), pyrogen-free saline (Thermo Fisher Scientific, North Ryde, New South Wales, Australia), Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, North Ryde, New South Wales, Australia), bovine serum albumin (BSA) (Sigma-Aldrich), alkaline phosphatase (AP) conjugated anti-mouse (Sigma-Aldrich), anti-rabbit and anti-human IgG (Sigma-Aldrich), β2GPI (Hematologic Technologies, Essex Junction, Vermont), isotype control rabbit polyclonal IgG (BD Biosciences) and red blood cell lysis buffer (eBioscience, San Diego, California). Triple-Pure High Impact Zirconium 1.5 mm beads (Benchmark Scientific, NJ) Affinity purified murine IgG2, anti-β2GPI monoclonal antibody and affinity purified polyclonal rabbit anti-β2GPI antibody were produced as previously described^{22 (link), 23} .
PE-rat anti-mouse CD41, APC-Cy7 rat anti-mouse CD45, BV510 hamster anti-mouse CD3e, PerCP-Cy5.5 rat anti-mouse CD4, PE-Cy7 rat anti-mouse CD8a, APC rat anti-mouse CD19, V450 rat anti-mouse LY-6G and LY-6C, PE rat anti-mouse CD11b and mouse Fc block^TM were purchased from BD Biosciences and used at 0.2 mg/mL.

Splenocytes (2 × 10⁶ per test) harvested from mouse spleen were stimulated with 2 μg/ml pooled peptides of SARS-CoV-2 RBD protein (15-mer peptides with 11–amino acid overlap covering the entire RBD protein; GenScript; refer to Supplementary Table S2) for an 18-h incubation in a CO₂ incubator. Protein transport inhibitors (BD GolgiPlug; BD Biosciences) were added and incubated for 6 h. Cells were then stained for flow cytometry analysis using the following antibodies from BD Biosciences: FITC Rat Anti-Mouse CD4, PE-Cy7 Rat Anti-Mouse CD8a, and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit. Subsequently, cells were fixed and permeated by using Fixation/Permeabilization Solution Kit, and further stained with APC Rat Anti-Mouse IFN-γ and PE Rat Anti-mouse IL-2. Finally, the samples were measured using a BD LSRFortessa X-20 Flow Cytometer (BD), with the data were analyzed by FlowJo V10.6.0. The gating strategy for detecting T cell subsets is shown in Supplementary Figure S1.