Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children Research Institute, Canada.
Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children Research Institute, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada.
J Immunol Methods. 2019 Apr;467:1-11. doi: 10.1016/j.jim.2019.01.002. Epub 2019 Feb 13.
New procedures are required to optimize the use of blood samples to study different cell types. The purification of neutrophils and T cells from the same blood sample is not commonly described. We have previously used PolymorphPrep™ (P) or LymphoPrep™ (L) for purifying neutrophils or T cells, respectively. In this study, we describe a new method for purifying both of these cells using P and L from the same sample, and methodological considerations required to obtain consistent Th17 differentiation results. For T cell studies, we first isolated mononuclear cells from peripheral blood of healthy humans using either P alone, L alone or sequential isolation with P and then L (P + L). CD3 lymphocytes comprise up to 73% of peripheral blood mononuclear cells (PBMCs) obtained by sequential isolation, with 29% and 36% for P and L, respectively. T lymphocyte subsets, Th1, Th17 or double-positive (Th17/1), were then amplified. Four days of amplification culture after isolation by P alone led to over-expression of Th17/1 cells and of Th17 cells in comparison to cells isolated by L or by sequential P + L. Th17/1 cells comprised 11.0 ± 6.8% (P alone) vs 1.2 ± 0.28% (L alone) vs 0.45 ± 0.11% (P + L) and Th17 cells comprised 2.8 ± 0.4% (P alone) 0.88 ± 0.15% (L alone) vs 0.86 ± 0.14% (P + L). As the second step, we examined T cell purification and differentiation. A higher purity of 97.1 ± 0.44% naïve CD4 T cell was reached after P + L followed by immunomagnetic bead sorting in comparison to 70 ± 9.3% (L) vs 21.0 ± 8.5% (P). These cells grew well in the density range of 25, 000 to 100, 000 cells per well in 96-well plates during Th17 cell differentiation; higher or lower cell density did not support Th17 cell differentiation. Lastly, to investigate the effect of estrogen on Th17 cell differentiation, serum-free AIM V medium without phenol red was chosen to minimize the hormonal effects of the medium. We found that exogenous estrogen (1 nM) inhibited Th17 cell differentiation in this medium. Taken together, we devised a method to isolate both neutrophils and T cells from the same blood sample and show that high PBMC purity, selected culture medium and an optimal cell density of the initial cell culture produced the most robust and consistent results for Th17 differentiation.
需要新的程序来优化血液样本的使用，以研究不同的细胞类型。从同一样本中纯化中性粒细胞和 T 细胞的方法并不常见。我们之前分别使用 PolymorphPrep（P）或 LymphoPrep（L）来纯化中性粒细胞或 T 细胞。在这项研究中，我们描述了一种从同一样本中使用 P 和 L 同时纯化这两种细胞的新方法，以及获得一致 Th17 分化结果所需的方法学考虑因素。对于 T 细胞研究，我们首先使用 P 单独、L 单独或 P 后 L（P+L）的顺序分离，从健康人外周血中分离单核细胞。通过顺序分离获得的外周血单个核细胞（PBMC）中 CD3 淋巴细胞最多可达 73%，P 和 L 分别为 29%和 36%。然后扩增 Th1、Th17 或双阳性（Th17/1）T 淋巴细胞亚群。单独使用 P 分离后，经过 4 天的扩增培养，导致 Th17/1 细胞和 Th17 细胞的过度表达，与使用 L 或 P+L 分离的细胞相比。Th17/1 细胞占 11.0±6.8%（P 单独）比 1.2±0.28%（L 单独）比 0.45±0.11%（P+L），Th17 细胞占 2.8±0.4%（P 单独）0.88±0.15%（L 单独）比 0.86±0.14%（P+L）。作为第二步，我们检查了 T 细胞的纯化和分化。与单独使用 L（70±9.3%）相比，P+L 后使用免疫磁珠分选，可获得更高纯度的 97.1±0.44%幼稚 CD4 T 细胞，21.0±8.5%（P）。这些细胞在密度范围为 25,000 至 100,000 个细胞/孔的 96 孔板中在 Th17 细胞分化期间生长良好；更高或更低的细胞密度不支持 Th17 细胞分化。最后，为了研究雌激素对 Th17 细胞分化的影响，我们选择不含酚红的无血清 AIM V 培养基，以最大限度地减少培养基的激素影响。我们发现，外源性雌激素（1 nM）抑制了该培养基中的 Th17 细胞分化。总之，我们设计了一种从同一样本中分离中性粒细胞和 T 细胞的方法，并表明高 PBMC 纯度、选择的培养基和初始细胞培养的最佳细胞密度为 Th17 分化产生了最稳健和一致的结果。
