IMPORTANCE OF CALIBRATION IN FLOW CYTOMETRY ANALYSIS FOR HUMAN ADIPOSE TISSUE – DERIVED STEM CELL CHARACTERIZATIONAbstract
Human adipose tissue-derived stem cells (hADSCs) are promising for cellular therapy as they are multipotent, which consistently express high CD90, CD105, CD73 and low hematopoietic surface markers. Flow cytometry is a routine technology that is used in most laboratories and can be used to study hADSC characterization, but the instrument needs strict calibration to assure the quality of the results. However, for rarely used fluorophores, calibration is often neglected. This study aimed to compare calibrated and non-calibrated fluorescence (FL) in flow cytometry performances. hADSCs were isolated from lipoaspirate and cultured in human platelet lysate supplemented medium. Passaged cells were used for characterization. Cells stained with positive (CD90-FITC, CD105-PerCP Cy5.5, CD73-APC) and negative (CD34, CD45, CD11b, CD19, HLA-DR)-PE marker cocktails were subjected to flow cytometry analysis. In the instrument, FITC, PE, PerCP- Cy5.5, and APC were detected by FL1, FL2, FL3, and FL4 respectively. Routine calibration of FL1, FL2, and FL3 was done, except for FL4. Therefore, data from calibrated and non-calibrated FL4 were analyzed. hADSCs strongly and consistently expressed CD90, CD105, and negative surface markers at averages of 94.6%, 90.5%, and 2.8% respectively, hence in line with ISCT guidelines; in contrast to CD73 (FL4), whose average was 42%. Characterization of calibrated FL4 showed very good results on CD73 (above 94%), but not in non calibrated FL4, which showed inconsistent results, as values ranged between 0% – 82%. In conclusion, routine calibration of FL1, FL2, FL3, and FL4 is important to get a reliable result.
P. Sari, E. Luviah, R. Y. Purwoko and J. A. Pawitan*
Department of Histology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
16 April, 2017
12 June, 2017
29 June, 2017
01 December, 2017