Purification of Megakaryocytes for flow cytometric analysis

Abstract

Objectives: Megakaryocytes (MKs) represent only a limited cell number in normal bone marrow, which hampers the analysis of MKs by flow cytometric technique. To optimize the usage of flow cytometry for the analysis of megakaryopoiesis, we established a simple method to enrich MKs. Methods: Bone marrow tissues were taken from mice (n=5) and made into cell suspensions. The cell suspension was processed by velocity sedimentation with a continuous BSA solution (3%-6%) for 1h at room temperature from top to bottom of the gradient to purify MKs. Each layer (1 ml) gradient from top to bottom was collected and the number of MKs and other cells was counted under a microscope. The enriched MKs gradients were located in the lower 5 ml between layer 7 and layer 11 following sedimentation. They were then collected, centrifuged and labeled by antibody anti-CD41-FITC, and their DNA was also labeled by propidium iodide. Then, cells were performed isotype antibody labeling instead of CD41 for flow cytometric analysis. Another group underwent the same procedures at the beginning of cell suspension. Results: Under the microscope, The MKs were enriched mainly in the lower 7~11 layers, while other small cells were largely enriched in the upper 1~6 layer following sedimentation. The ploidy analysis by flow cytometric analysis showed that there was a typical peak which can represent various DNA content in the enriched MKs group, while the peak was not typical in the group without MK isolation. Conclusions: The results showed that the purification of MKs was effective in this experimental condition, providing a possibility to analyze megakaryopoiesis by flow cytometry instrument.