Quantification of leukocyte migration: improvement of a method.
Quantification of leukocyte migration: improvement of a method.
复制标题
白细胞迁移的定量：方法的改进。
DOI：
10.3109/08820139609059290
复制
发表时间：
1996
影响因子：
2.8
通讯作者：
W. Hörl
中科院分区：
文献类型：
--
作者：
G. Sunder;R. Hofbauer;G. Sengoelge;W. Hörl
18种不同的通透膜支架与正常供体来源的中性粒细胞在24孔双室系统的上部腔中孵育，其中有和没有融合内皮细胞单层。为了研究白细胞的跨膜或跨内皮迁移，用化学诱导剂刺激白细胞，或用IL-1激活内皮细胞。共培养后，丢弃构建上腔的膜支撑物。使用这种技术，迁移到下室的白细胞被暴露在荧光染料Calcein AM中，而不需要额外的洗涤或转移步骤。以钙黄绿素AM标记的白细胞稀释系列为标准，计算机辅助测定绝对细胞计数。钙黄绿素AM作用于中性粒细胞的系列稀释液显示出重复性的线性荧光强度，且相对荧光强度与细胞计数显著相关(R2=0.974，p<；0.0001)。在18种膜载体中，只有一种适合我们建立的检测方法。用孔径为3 mm、孔密度为0.8×10(6)/cm2的聚对苯二甲酸乙二酯制成的膜具有最好的技术和光学性能。经FMLP或IL-1刺激的白细胞和内皮细胞的跨内皮迁移分别增加到7.2+/-1.8×10(5)PMN和5.1+/-0.7×10(5)PMN，与培养基中性粒细胞(0.6+/-0.2×10(5)PMN)相比。来自慢性肾功能衰竭患者的抗IL-1自身抗体可抑制IL-1诱导的中性粒细胞迁移(IL-1：100%的PMN迁移，抗IL-1抗体：39%的PMN迁移，对照抗体：84%的PMN迁移)。综上所述，建立了一种简单的测定跨膜和跨内皮细胞迁移的荧光分析方法。
Eighteen different permeable membrane supports with and without confluent endothelial cell monolayers were incubated with normal donor derived neutrophils in the upper chambers of a 24 multiwell double chamber system. In order to study transmembrane or transendothelial leukocyte migration leukocytes were stimulated by chemoattractants, or endothelial cells were activated by IL-1. After coincubation the membrane supports building the upper chambers were discarded. Using this technique, leukocytes that had migrated into the lower chamber were exposed to the fluorescent dye calcein AM without additional washing or transfer steps. Absolute cell counts were determined computer assisted using dilution series of calcein AM labeled leukocytes as standards. Serial dilutions of neutrophils exposed to calcein AM showed reproducible linear fluorescence intensity, and relative fluorescence intensity correlated significant with cell counts (r2 = 0.974, p < 0.0001). Out of 18 membrane supports only one was suitable for our assay set up. Best technical and optical performance was achieved with a membrane made of polyethylene terephtalate with a pore size of 3 mm at a pore density of 0.8 x 10(6)/cm2. Stimulation of leukocytes or endothelium by FMLP or IL-1 revealed an increase of transendothelial migration to 7.2 +/- 1.8 x 10(5) PMN and 5.1 +/- 0.7 x 10(5) PMN respectively if compared with medium (0.6 +/- 0.2 x 10(5) PMN). IL-1 induced migration of neutrophils was inhibited by anti IL-1 autoantibodies derived from chronic renal failure patients (IL-1: 100% of PMN migrated, anti IL-1 antibody: 39% of PMN migrated, control antibody: 84% of PMN migrated). In summary, a simple fluorimetric assay was established for the quantification of transmembrane and transendothelial leukocyte migration.
影响因子：
20.3
作者：
T. Carlos;J. Harlan
通讯作者：
T. Carlos;J. Harlan
透析过程中粒细胞 LAM-1 和 MAC-1 的调节——一项前瞻性、随机对照试验。
影响因子：
19.6
作者：
Himmelfarb,J;Zaoui,P;Hakim,R
通讯作者：
Hakim,R