Khalaf A N, Wolff-Vorbeck G, Bross K, Kerp L, Petersen K G
Abteilung Klinische Endokrinologie, Medizinische Universitätsklinik, Freiburg, Germany.
J Immunol Methods. 1993 Sep 27;165(1):121-5. doi: 10.1016/0022-1759(93)90113-l.
A method for labelling mouse spleen cells in situ is described. Spleen vessels were clamped before intrasplenic injection of a red-fluorescent cell dye (PKH-26). The labelling rate was 11.8% of all cells and 13.9% of B lymphocytes 30 min after injection as determined by FACS. 3 days later, the proportions of labelled cells were reduced to 7.4% (P < 0.01) and to 10.7% (P < 0.05), respectively. Only 10% of cells detected by FACS could be detected by fluorescence microscopy. Labelled cells were not found in peripheral lymph nodes 30 min after spleen injection, but were present 3 days later (FACS: 2.8% of all cells and 5.4% of B lymphocytes, P < 0.05 each), indicating migration of stained cells. Neither adverse nor toxic effects of in situ staining were observed. Isolated stained B lymphocytes from spleens of naive mice and from lymph nodes after immunisation with insulin showed proper function when tested in an ELISA-spot assay. The labelling procedure was used to follow splenic B lymphocytes producing natural anti-insulin antibody. These cells were found to be recruited for the early immune response in lymph nodes immunised with insulin.
本文描述了一种对小鼠脾细胞进行原位标记的方法。在脾内注射红色荧光细胞染料（PKH-26）之前，先夹住脾血管。注射30分钟后，通过流式细胞术测定，标记率为所有细胞的11.8%，B淋巴细胞的13.9%。3天后，标记细胞的比例分别降至7.4%（P<0.01）和10.7%（P<0.05）。通过流式细胞术检测到的细胞中，只有10%能用荧光显微镜检测到。脾注射30分钟后，外周淋巴结中未发现标记细胞，但3天后出现了标记细胞（流式细胞术：所有细胞的2.8%，B淋巴细胞的5.4%，P均<0.05），表明染色细胞发生了迁移。未观察到原位染色的不良或毒性作用。在用胰岛素免疫后，从幼稚小鼠脾脏和淋巴结中分离出的染色B淋巴细胞在酶联免疫斑点分析中检测时显示出正常功能。该标记程序用于追踪产生天然抗胰岛素抗体的脾B淋巴细胞。发现这些细胞被招募到用胰岛素免疫的淋巴结的早期免疫反应中。
