D.2 Reagents

D.2.1 Phosphate-buffered saline (PBS): pH 7.4.

D.2.2 Disodium ethylenediaminetetraacetic acid solution: 1%

D.2.3 Red blood cell lysis buffer.

D.2.4 Antibodies and isotype controls. For antibody storage conditions, please refer to product instructions.

D.3 Procedure

D.3.1 Sample preparation

Harvest single cells by centrifugation (D.1.2) at 300 g for 5 min. Discard the supernatant. Collect the cells and resuspend the cells. Determine the concentration of the viable cells in suspension, according to the method in Appendix A. Adjust the concentration of cell suspension to 5 × 106 cells per ml.

D.3.2 Cell transplantation

5.0 × 105 cells were injected into sublethally irradiated (200-300 cGy) NOD-SCID Il2rgnull mice at 6 to 8 weeks of age via the tail vein, setting up a blank control group that was injected with an equal volume of phosphate buffer to the cell suspension. The number of mice per group was ≥8.

D.3.3 Peripheral blood cell collection and antibody labelling

After 16 weeks of cell injection, collect the anticoagulated peripheral blood of transplanted mice, lyse the red blood cells, and label the human CD45 antibody, CD19 antibody, CD3 antibody, CD33 antibody, CD235a antibody or the corresponding isotype control, according to the method in Appendix A. After cell incubation and washing, perform flow cytometry (D.1.3) to analyse the percentage of human CD45+ cells, human CD45+ CD19+ cells, human CD45+ CD3+ cells, human CD45+ CD33+ cells and human CD45−CD235a+ cells.

D.3.4 Result analysis

Using flow cytometry software to analyse the results of flow cytometry, the percentage of human CD45+ cells in the PBMCs is not less than 5%.

Unlabelled PBSCs from transplanted mice were used as negative control 1, and antibody-labelled PBMCs from transplanted mice were used as biological control 2.

Firstly, according to the FSC and SSC, the target cell group 1 was gated, and the dead cells, platelets, red blood cells and cell fragments were excluded. According to the fluorescence intensity of negative control 1, the positive gate was delimited, and the positive cell group 2 was gated based on group 1.

Following the above gate setting principles, human CD45+ CD19+ cells, human CD45+ CD3+ cells, human CD45+ CD33+ cells and human CD45− CD235a+ cells were detected. The tested positive rate minus the positive rate of control 2 is the actual positive rate of the tube. If the value is greater than 1%, it is considered positive.