Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases

Abstract

Koistinen P. Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases. Scand J Clin Lab Invest 1987; 47: 709-714. Density gradient centrifugation on Lymphoprep as described by Boyum [ 11 was used for mononuclear cell separation of peripheral blood (PB) and bone marrow (BM) samples in healthy subjects and in haematological diseases. The cells were analysed morphologically from cytocentrifuge preparations stained with May-Griinwald-Giemsa. At least 200 cells per preparation were counted manually. The separation results of PB samples in healthy subjects were comparable with those described by Boyum. This method was also suitable for the separation of lymphocytes and blast cells in chronic lymphoproliferative diseases and in acute leukaemias, respectively. In other haematological diseases, however, the mononuclear cell fractions of PB samples and especially of BM samples were contaminated with myeloid and erythroid cells. Percoll gradients were used with success for the enrichment of myeloid BM cells and PB reticulocytes. However, absolutely pure myeloid cell fractions from different maturation stages could not be separated. The results indicate that cells to be investigated should be morphologically examined, to avoid erroneous interpretation of biochemical and functional activity of the cells.

Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer’s disease patients

“…In all cases peripheral blood samples were obtained after written informed consent of the patients or their relatives. A summary of demographic characteristics of all subjects enrolled in this study is reported in Table 1. Establishment of lymphoblastic cell lines was performed in our laboratory as previously described by infecting peripheral blood lymphocytes with the Epstein-Barr virus (Koistinen, 1987). Cells were grown in suspension in T flasks in an upright position, in approximately 8 mL of RPMI-1640 (Gibco, BRL) medium that contained 2 mM Lglutamine, 100 g/mL penicillin/streptomycin and, unless otherwise stated, 10 % (v/v) fetal bovine serum (FBS) and maintained in a humidified 5% CO 2 incubator at 37 ºC.…”

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia

“…Establishment of lymphoblastic cell lines was performed in our laboratory as previously described (Ibarreta et al, 1998) by infecting peripheral blood lymphocytes with the Epstein Barr virus (EBV) (Koistinen, 1987). Cells were grown in suspension in T flasks in an upright position, in approximately 10 ml of RPMI-1640 (Invitrogen) medium that contained 2 mM L-glutamine, 100 mg/ml penicillin/streptomycin and, unless otherwise stated, 10 % (v/v) fetal bovine serum (FBS) and maintained in a humidified 5% CO 2 incubator at 37 o C. Fluid was routinely changed every two days by removing the medium above the settled cells and replacing it with an equal volume of fresh medium.…”

Human splenic polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) are strategically located immune regulatory cells in cancer

“…Nevertheless, splenic neutrophils isolated from the same density fraction with normal-density blood neutrophils were functionally as suppressive as low-density PMN-MDSC. Not only the level of myeloid maturation correlates with granulocytes' density but also a cell's density can be altered under the influence of various physical forces as they dynamically travel through various compartments [24,25]. Compared to the peripheral blood leukocytes, the splenocytes expressed higher levels of chemoattractants, S100A9 and S100A8, which may have contributed to the accumulation of PMN-MDSC in the spleen.…”