Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14⁺ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14⁺ (88 %), MHC-II⁺ (91.5 %) and CD11b⁺ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

使用健康绵羊（n  = 3）和山羊（n  = 3）的外周血建立一种同时分离外周血单核细胞（PBMC）和中性粒细胞的有效方法，并标准化用于纯化单核细胞和产生单核细胞的方案巨噬细胞（MDM）。与使用相比，通过密度梯度处理血沉棕黄层和红细胞层（RBC）的混合物时，在这两个物种中，PBMC的数量都大大丰富，具有更高的纯度和通过流式细胞仪测定的细胞数量只是血沉棕黄层（p <0.05）。嗜中性粒细胞可以随后从位于PBMCs组分下面的层中分离出来，其纯度明显高于通过流式细胞仪测定的纯度，高于使用无密度梯度的实验获得的纯度（<60％）（p  <0.05）。该技术将允许从相同的血液样本中分离出两个细胞群。使用免疫磁柱从PBMC中纯化出单核细胞CD14 ⁺细胞的纯细胞群，可提供17％（nº单核细胞/nºPBMC）的产量和高百分比的CD14 ⁺（88％）MHC-II ⁺（91.5％）和CD11b ⁺（94％）通过流式细胞仪确定。另一方面，基于前者的粘附能力从PBMC中经典且非昂贵地纯化单核细胞，使得单核细胞的产量显着降低（4.6％），表面标志物表达的百分比分别降至35％，65％分别为55％和55％（p < 0.001），表明分离出混合细胞群。与不添加或使用自体血清的情况相比，以25至125 ng / mL的浓度向培养物中添加GM-CSF所产生的MDM数量成比例地增加了5％至20％。但是，通过粘附方法纯化单核细胞比通过两种物种的免疫磁柱分离的单核细胞的产率更高（p <0.001）。在这项研究的条件下，使用密度梯度离心可以从同一份血液中同时纯化PBMC和嗜中性粒细胞，两个种群的纯度都很高。单核细胞的分离随后可以通过两种不同的方法来实现，即基于免疫磁柱或粘附。两种方法之间的偏好取决于实验的必要性，具有高纯度单核细胞的初始样品或所需的MDM最终种群。