Ebioscience 1x rbc lysis buffer

Whole blood was collected into EDTA coated tube. For 1 ml whole blood, 9 ml of eBioscience™ 1X RBC Lysis Buffer (Invitrogen, Waltham, MA, USA) was added for red blood cells (RBCs) lysis, after 10 min, cells were centrifuged and resuspended in flowcytometry buffer (3% Heat inactivated FCS, 1 mM EDTA, 10 mM HEPES, 0.09% NA acid), 2 million cells were blocked with 2 µl Mouse BD FcBlock™ (BD Biosciences) for 5 min on ice, cells were resuspended in antibody cocktail for 20 min after centrifugation, then, washed twice with cold PBS and stained with FITC Annexin V kit (Biolegend, San Diego, CA, USA) and 7-AAD (Invitrogen, Waltham, MA, USA) according to manufacturer’s instructions. Antibodies used in the antibody cocktail can be found in Supplementary Table 1. Samples were acquired by using BD FACSLyric™ flow cytometry (BD Biosciences). UltraComp eBeads™ Compensation Beads (Invitrogen, Waltham, MA, USA) were used to set up the compensation. Fluorescence minus one (FMO) samples were used to identify negative population for each antibody. Data analyze were performed by using FlowJo software (version 10.8; Tree Star, Ashland, USA).

In a separate Tu-2449SC subcutaneous tumor model experiment, mice from each assigned group that showed measurable tumor growth were sacrificed at day 39 post tumor implantation. Tumors were excised from the skin and minced into small pieces in HBSS (ThermoFisher Scientific, # 14175095) and single-cell suspensions were obtained using gentleMACS™ C Tubes with gentleMACS™ Dissociator (Miltenyi Biotec, # 130-096-334 and # 130-093-235), and enzymatic solution containing 50 µg/mL final Liberase™ TM (Sigma-Aldrich, # 5401119001) and 200 U/mL final Deoxyribonuclease I (Sigma Aldrich, # D5025). After filtration through a 70 µm cell strainer and washing, erythrocytes were removed following incubation with eBioscience 1X RBC Lysis Buffer (ThermoFisher Scientific, # 00-4300-54). Viable cell counts were determined using a Cellometer K2 (Nexcelom Bioscience). For subsequent flow cytometry analyses, cells were first incubated with TruStain FcX antibody (BioLegend, # 101320) and Zombie UV (BioLegend, # 423108) before being subjected to surface staining with fluorochrome-conjugated antibodies against mouse CD45 (BioLegend, # 103132), CD3ɛ (BD Biosciences, # 563565), CD4 (BD Biosciences, # 564922) and CD8α (BioLegend, # 100759). Acquisition was conducted on an LSRFortessa X-20 flow cytometer and analysis by using FlowJo software (version 10) with flowAI (1.7) plugin as described [52 (link)].

To obtain single-cell suspension, livers were minced by a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach). Then, red blood cells were lysed (eBioscience™ 1X RBC Lysis Buffer, Thermo Fisher Scientific) and IHLs (intrahepatic lymphocytes) were separated using a 40%/70% Percoll (GE Healthcare) gradient. Subsequently, lymphocytes were stained with anti-CD3, anti-CD4, anti-CD8, anti-Ki-67, anti-B220, anti-Foxp3, and anti-CD62L antibodies. All image acquisitions were performed with an LSRII SORP interfaced with DIVA software (BD Biosciences) as described previously [16 (link), 19 (link)].

The whole lungs collected at 30 DPI¹ were dissociated into a single cell suspension using gentleMACS^™ Octo Dissociator (Miltenyi Biotec) and Mouse Lung Dissociation Kit, mouse (Miltenyi Biotec) as per manufacturer’s instructions. The single cell suspension was then treated with eBioscience^™ 1X RBC Lysis Buffer (ThermoFisher Scientific) to remove erythrocytes. The treated cells were then washed twice with PBS and resuspended in eBioscience^™ Flow Cytometry Staining Buffer (ThermoFisher Scientific) containing 1:50 Mouse BD Fc Block^™ (BD biosciences) antibody and incubated at room temperature for 15 mins, followed by staining with anti-CD3, CD8, CD44, CD69, CD103 (BD biosciences) and SARS-CoV-2 spike tetramer (VNFNFNGL, NIH core tetramer) for 15 mins at room temperature in the dark. The cells were washed twice with eBioscience^™ Flow Cytometry Staining Buffer (ThermoFisher Scientific) and fixed by adding 10% formaldehyde (5% final concentration in staining buffer) and incubating for 48hrs at 4°C. Fixed cells were washed twice with staining buffer and filtered through a 100μm cell strainer (Falcon) to remove clumped cells. Filtered single cell suspensions were then analyzed using the Gallios Flow Cytometer (Beckman Coulter) and FlowJo software.