DST-NRF Centre of Excellence for Biomedical Tuberculosis Research; Stellenbosch University, Cape Town, South Africa.
South African Medical Research Council Centre for Tuberculosis Research; Stellenbosch University, Cape Town, South Africa.
PLoS One. 2019 Mar 20;14(3):e0213832. doi: 10.1371/journal.pone.0213832. eCollection 2019.
This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection-in which B-cells remain "untouched" in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection-in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays.
本文介绍了使用各种 Miltenyi 磁性激活细胞分选 (MACS) 珠分离试剂盒从全血中分离纯 B 细胞群体的步骤。对于研究 B 细胞功能能力的研究，这样的群体是至关重要的，因为其他细胞类型的存在可能通过细胞间相互作用或通过分泌几种可溶性分子对 B 细胞功能产生间接影响。B 细胞可以通过两种主要方法进行分离：1）阴性选择-其中 B 细胞在其自然状态下“未受触动”；这是有利的，因为 B 细胞在这个过程中可能仍然保持功能不变。2）阳性选择-其中 B 细胞被标记并从样本中主动去除。我们使用了三种阴性 B 细胞分离试剂盒以及 Miltenyi 的阳性 B 细胞分离试剂盒，并比较了每种分离后 B 细胞群体的纯度。用所有测试的分离技术分离出的 B 细胞群体都存在血小板污染，这是决定性的发现。这些结果表明，目前可用的 MACS B 细胞分离试剂盒无法有效地产生纯 B 细胞群体，从中可以得出具体的结论。因此，我们建议使用细胞分选作为分离纯 B 细胞的首选方法，用于下游功能测定。
