Bailey Tanya, Stark Sarah, Grant Angela, Hartnett Christopher, Tsang Monica, Kalyuzhny Alexander
Department of Immunocytochemistry, R&D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413, USA.
J Immunol Methods. 2002 Dec 15;270(2):171-82. doi: 10.1016/s0022-1759(02)00297-1.
Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.
使用冷冻保存的外周血单个核细胞（PBMC），而非在不同日期从同一供体采集的新鲜细胞，克服了这些细胞对激活剂敏感性的变异性。为了解冷冻保存的影响，对来自八位健康供体的冷冻PBMC进行研究，使用酶联免疫斑点分析（ELISPOT分析）来释放包括白细胞介素-1β（IL-1β）、白细胞介素-2（IL-2）、白细胞介素-4（IL-4）、白细胞介素-6（IL-6）、白细胞介素-13（IL-13）、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）在内的辅助性T细胞1（TH1）或辅助性T细胞2（TH2）细胞因子。使用三种PBMC浓度（5×10⁶、5×10⁵和5×10⁴个细胞/毫升）来确定斑点形成细胞（SFC）的数量。发现来自所有八位供体的PBMC在冻融后保留了释放TH1或TH2细胞因子的功能能力。当PBMC以5×10⁶或5×10⁵个细胞/毫升的浓度使用时，对于大多数供体而言，孔中与IL-1β、IL-2、IL-6和TNF-α相关的斑点密度似乎过高，使得SFC定量变得困难或不可能。相反，浓度为5×10⁴的PBMC产生了清晰且可定量的斑点。当PBMC以5×10⁶的浓度使用时，与IL-4和IL-13释放相关的斑点密度似乎最适合SFC定量，而在5×10⁵个细胞/毫升时斑点密度非常低，在5×10⁴个细胞/毫升浓度组中则不存在。除了IFN-γ和IL-2细胞因子外，未发现不同细胞因子释放水平之间的关系，这表明具有高IFN-γ反应的冷冻保存PBMC可能也具有高IL-2反应。我们的结果表明，一种细胞因子的释放水平可能无法通过了解另一种细胞因子的水平可靠地预测。这意味着有必要在广泛的浓度范围内测试冷冻保存的PBMC，以确定一种最适合产生清晰且可定量斑点的浓度。
