使用不连续Percoll稀释法从猫血液中分离中性粒细胞。

Neutrophil isolation from feline blood using discontinuous Percoll dilutions.

作者信息

Sursal Neslihan, Cakmak Ayse, Yildiz Kader

出版信息

Tierarztl Prax Ausg K Kleintiere Heimtiere. 2018 Dec;46(6):399-402. doi: 10.1055/s-0038-1677404. Epub 2019 Jan 18.

DOI:10.1055/s-0038-1677404

PMID:30658367

Abstract

OBJECTIVE

Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients.

MATERIAL AND METHODS

Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63 %, 54 % and 45 %). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72-63 % Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils.

RESULTS

The mean PMN number was 22 x 10 per ml (minimum - maximum: 20-26 x 10ml). Neutrophil homogeneity was > 95 % in the cell suspensions. The viability of isolated neutrophils was > 98 %. The technique did not result in neutrophil activation.

CONCLUSION AND CLINICAL RELEVANCE

Discontinuous Percoll gradients (72 %, 63 %, 54 % and 45 %) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.

摘要

目的

一些研究已开展从猫血液中体外分离中性粒细胞的工作。这些研究的主要限制因素是在不引发可能危及生命的并发症的情况下所能采集的血液量较少。在本研究中，我们尝试使用不连续的 Percoll 梯度从猫静脉血样本中分离中性粒细胞。

材料与方法

从临床健康的成年猫的头静脉采集血液。将血样铺在 Percoll 稀释液（72%、63%、54%和 45%）上。离心后，猫多形核白细胞（PMN）在 72% - 63%的 Percoll 稀释液之间聚集成一条带。使用光学显微镜计数来计算细胞总数。用 Diff - Quik 染色后通过显微镜观察确定中性粒细胞的百分比。用 0.01%台盼蓝试验评估中性粒细胞的活力。根据分离出的中性粒细胞完整的细胞形态来确定其活化情况。