SARS-CoV-2 stabilized spike glycoprotein (BEI Resources, NR-53257) (57 (link)) was coated at a concentration of 2 μg/ml in carbonate buffer on 96-well MultiScreen HTS IP Filter plates (Millipore) overnight at 4°C. Plates were blocked with complete RPMI (10% heat-inactivated FBS, 1% Penicillin/Streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 55 μM 2-mercaptoethanol) for 4 hr at 37°C. Bone marrow cells were isolated from the left femur + tibia of mice infected with SARS-CoV-2. Red blood cells were lysed with RBC Lysis Buffer (BioLegend) for 2 min. Cells were resuspended in complete RPMI and plated in duplicate at three dilutions (1/5, 1/10, and 1/20 of total bone marrow cells) for 20 hr at 37°C. Plates were washed six times with PBS-T (0.01% Tween-20), followed by incubation with anti-mouse IgG-Alkaline Phosphatase (Southern Biotech, 1030-04) in PBS with 0.5% BSA for 2 hr at room temperature. Plates were then washed three times with PBS-T and three times with PBS. Spots were developed with Vector® Blue Substrate Kit (Vector Laboratories, SK-5300) and imaged with an ImmunoSpot analyzer (Cellular Technology Limited). Spots were counted manually by a blinded investigator.