Cd3 apc clone hit3a

To assess the number of myeloid cells from plasma incubation experiments, cells were stained with the following panel: CD3-APC (clone HIT3a), CD19-APC (clone HIB19), CD56-APC (clone 5.1H11), CD14-FITC (clone M5E2), CD15-AF700 (clone HI98), CD11b-PE-Cy7 (clone ICRF44), CD34-BV650 (clone 561), and CD38-PE/Cy5 (clone HIT2) (BioLegend). After staining, cells were resuspended in FACS buffer with 2% CountBright beads (Invitrogen) to allow determination of absolute counts during analysis. Flow cytometry data were acquired on a Cytoflex LX (Beckman Coulter) and analyzed using FlowJo v10.1.

For T cell mixing experiment, Flu-m1 TCR expressing Jurkat T cells were labeled by CellTrace Violet dye (#C34571, Thermofisher) according to manufacturer’s protocol. Violet labeled Flu-m1 TCR+ T cells were mixed with NY-ESO-1 TCR+ T cells at diverse ratios including 1:1, 1:10, 1:100, 1:1000. The mixed T cells were incubated with 40uL concentrated HLA-A2-Flu antigen displayed GFP viruses for 2 hr at 37°C. T cells were stained with CD3-APC (clone HIT3a, BioLegend) antibody, washed twice, and subjected to flow cytometry. For B cell mixing experiment, Spike-RBD BCR expressing Ramos B cells were labeled by CellTrace Violet dye and mixed with HPV-L2 BCR expressing Ramos B cells at diverse ratios including 1:1, 1:10, 1:100, 1:1000. The mixed cells were incubated with 40uL concentrated RBD-antigen displayed GFP viruses for 2hr at 37°C. B cells were stained with IgG-PE-Cy7 antibody (clone G18–145, BD Biosciences), washed twice, and subjected to flow cytometry. The metrics are calculated below:
$$\text{Sensitivity}=\text{percentage}\text{of}\text{GFP+}\text{on-target}\text{cells}\text{among}\text{total}\text{on-target}\text{cells}$$
$$\text{Specificity}=1-\left(\text{percentage}\text{of}\text{GFP+}\text{off-target}\text{cells}\text{among}\text{total}\text{off-target}\text{cells}\right)$$
$$\text{Signal-to-noise}\text{ratio}=(\text{percentage}\text{of}\text{GFP+}\text{on-target}\text{cells}\text{among}\text{total}\text{on-target cells})/\left(\text{percentage}\text{of}\text{GFP+}\text{off-target}\text{cells}\text{among}\text{total}\text{off-target}\text{cells}\right)$$