X vivo 10 serum free hematopoietic cell medium

All samples reached the lab within 1 hour of collection and were either processed immediately or frozen for later pooling. To each sample an equivalent volume of X-VIVO^TM 10 Serum-free Hematopoietic Cell Medium (Lonza) was added to maintain cell viability. Owing to the low concentration of cells in the CSF, each sample was first concentrated by centrifugation at 300g for 10 min, the supernatant removed leaving approximately a 200 μl volume. A manual cell count was completed on the concentrated sample using a Neubauer haemocytometer and cell viability assessed using Trypan Blue exclusion dye. The cell suspension was then centrifuged at 300g for another 10 min to further concentrate the sample to a final volume of 32 μl ready to be loaded onto a 10x Chromium Single Cell Controller using the Chromium Single Cell Gene Expression 3′ v2 kit (10x Genomics). Sequencing was completed on an Illumina HiSeq 4000 at the Cancer Research Institute, Cambridge University sequencing hub using locally optimized protocols and settings.

The ELISpot assay was performed using precoated human SARS-CoV-2-specific IFNγ ELISPOT kits (AutoImmun Diagnostika). Peripheral blood was collected, PBMCs were isolated and erythrocytes depleted (RBD-Lyse Buffer Life Technologies). PBMCs were counted and resuspended in X-VIVO medium (X-VIVO TM-10 Serum-free Hematopoietic Cell Medium, Lonza).
In brief, a total of 2 × 10⁵ PBMCs were incubated in duplicates with X-VIVO as a negative control, pokeweed mitogen (AutoImmun Diagnostika GmbH) as a positive control, and 15–20mer peptide pools for SARS-CoV-2 and PanCorona for the four non-SARS human coronaviruses 229E, HKU1, NL63, and OC43 (AutoImmun Diagnostika GmbH) as a control of possible cellular cross-reactive responses. After incubation at 37°C and 5% CO₂ for 20 h, plates were washed with washing buffer (AutoImmun Diagnostika GmbH) and stained with the kit-specific reagents according to the protocol of the manufacturer. Spots were counted using an automated AID ELISPOT reader system (AutoImmun Diagnostika GmbH).
The stimulation index (SI) was calculated by dividing the mean spot numbers in the antigen-specific wells with the mean spot numbers of the negative control. A test was assessed as negative with an SI <2 and positive with an SI ≥2.