2 time volume of PBS/EDTA buffer (including 0.5% of HSA: Human Serum Albumin) was added into 50-120ml of PBSPC obtained in 1 to 2 collections and the supernatant was removed after centrifuge. The volume was adjusted to 90-100ml and then 7.5ml of CliniMACS CD34 reagent was added. It was put into a rotator (25rpm) at 19-25℃ and cultured for 30 minutes, then washed with PBS/EDTA buffer twice. The final volume was made to be 100 to 300ml.
The above PBSPC leukapheresis products were connected with CliniMACS. "CD34 selection 1" programme was selected and the purification of CD34+ cells would start automatically. 42 to 45ml of final product was retained. The final purified CD34+ products were infused to 5 patients immediately after separation, while the other 22 purified products were stored in liquid nitrogen at -196oC after sustained programmed cryopreservation.
1.4 Blood cell counting and flow cytometry
The final purified product were diluted with the ratio of 1:20 for original solution to PBSPC 1:20. The cells were counted with cytometer ( CELL-DYN 1500, Abbott) and 50ul of the diluted solution was added into different tubes and then different McAbs labeled by fluorescent-marks such as CD45-Fitc/CD34-PE、CD3-Pc5/CD4-Fitc/CD8-PE、CD3-Fitc/CD19-PE、CD3-Fitc/CD56-PE etc. (IMMUNOTECH)and propidium iodide(PI ......
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