Nh4cl based erythrocyte lysing solution

A four-color flow cytometry panel was designed using a commercial CD19 CAR Detection Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). The reagent consists of a biotinylated CD19 antigen that specifically binds CD19-targeted CARs. In a second incubation step the biotin-labeled CAR T cells are then stained with a fluorochrome-conjugated anti-biotin antibody.
In brief, 200 µl whole blood were treated for 10 min with 2 ml of NH₄Cl-based erythrocyte lysing solution (Beckman Coulter, Krefeld, Germany) and washed with PBS, containing 0.5% HSA. After removal of the supernatant down to 200 µl, cells were resuspended and 100 µl were transferred to a new flow cytometry tube. Following 15 min of incubation with 1 µl CD19 CAR Detection Reagent, cells were washed twice and incubated for 15 min with 1 µl Anti-Biotin-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 µl 7-AAD, 5 µl CD3-APC, and 5 µl CD45-KrO (all purchased from Beckman Coulter Immunotech, Marseille, France). After a final washing step, cells were acquired on a NAVIOS flow cytometer (Beckman Coulter, Krefeld, Germany). Cellular debris was excluded based on light scatter properties and CAR T cells were defined as 7-AAD^-/CD45⁺/mononuclear cells/CD3⁺/CD19 CAR⁺ (Supplementary Figure 1).