Facsymphony a5 analyser

Threefold serially diluted recombinant human ACE2‐Fc (Acro Biosystems) or twofold serially diluted ACE2‐Bite and control supernatants were incubated with GFP‐encoding SARS‐CoV‐2 Spike‐pseudotyped viruses with 0.2 multiplicity of infection (MOI) for 1 h at 37°C degrees. The mixtures were subsequently added to ACE2+ 293 cells, which were previously stained for their ACE2 expressions and confirmed ~%100 positive before neutralisation assays, for 72 h after which cells were collected, washed with FACS buffer (1×PBS + 2% FBS) and analysed by flow cytometry using a BD FACSymphony A5 analyser. Cells not expressing GFP were used to define the boundaries between non‐infected and infected cell populations. Per cent infection was normalised for samples derived from cells infected with SARS‐CoV‐2 pseudotyped virus in the absence of ACE2‐Fc or ACE2‐Bite.

Cells were resuspended in staining buffer (PBS + 2% FBS) and incubated with fluorochrome‐conjugated antibodies for 30 min at 4°C. CD8 T cells were identified with CD8‐Pacific Blue antibody (Biolegend). Activation of CD8 CAR‐T cells was determined with CD25 staining using the CD25‐APC antibody (Biolegend). CAR expressions of ACE2 CAR and anti‐Spike CAR and ACE2 expression of ACE2‐293 cells were determined with SARS‐CoV‐2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti‐mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression. CAR expression of anti‐CD19 CAR was determined with Human CD19 (20–291) Protein, Fc Tag, low endotoxin (Super affinity) (Acro) followed by a secondary staining with APC conjugated anti‐human IgG Fc Antibody (Biolegend) and RFP expression. For cytotoxicity assay analysis, stably Spike protein‐expressing T2 and 293 cell lines were identified with GFP marker. For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2/ACEH Protein, Fc, Avitag (Acro Biosystems) and then stained with APC anti‐human IgG Fc Antibody clone HP6017 (Biolegend). Samples were acquired on a BD FACSymphony A5 analyser, and data were analysed using FlowJo (BD Biosciences).