Nakagawara A, Nathan C F
J Immunol Methods. 1983 Jan 28;56(2):261-8. doi: 10.1016/0022-1759(83)90418-0.
A simple method was devised for counting small numbers (10(4)-10(6)) of adherent mononuclear phagocytes, including populations containing multinucleated giant cells, which often arise during cultivation of human blood monocytes. Coverslips with adherent cells were transferred into small volumes (50-200 microliters) of 0.1 M citric acid, pH 2.2, containing 0.05% naphthol blue black and 1.0% of either Triton X-100 or Cetavlon. Triton X-100 was adequate for use with monocytes and macrophages from early cultures. However, Cetavlon was preferable for use with older cultures of adherent human mononuclear cells in order to prevent aggregation of the nuclei from giant cells. When multinucleated cells were present, separately stained coverslips were inspected to determine the mean number of nuclei per cell. This value, together with the number of nuclei per coverslip, permitted calculation of the number of cells per coverslip. The latter value is not readily derived from measurements of protein or DNA content in populations containing multinucleated giant cells. This counting method was simpler and more sensitive than several previously reported methods for enumerating adherent macrophages.
设计了一种简单的方法来计数少量（10⁴ - 10⁶）贴壁单核吞噬细胞，包括含有多核巨细胞的细胞群体，多核巨细胞常在人血单核细胞培养过程中出现。将带有贴壁细胞的盖玻片转移到小体积（50 - 200微升）pH 2.2的0.1 M柠檬酸中，其中含有0.05%萘酚蓝黑和1.0%的Triton X - 100或西他氯铵。Triton X - 100适用于早期培养的单核细胞和巨噬细胞。然而，西他氯铵更适合用于贴壁人单核细胞的较老培养物，以防止巨细胞核聚集。当存在多核细胞时，检查单独染色的盖玻片以确定每个细胞的平均核数。该值与每个盖玻片的核数一起，可计算出每个盖玻片的细胞数。在含有多核巨细胞的群体中，后者的值不易从蛋白质或DNA含量的测量中得出。这种计数方法比之前报道的几种计数贴壁巨噬细胞的方法更简单、更灵敏。
