Jiao H, Soejima Y, Ohe Y, Miura K, Tamura T, Saijo N
Department of Oral Surgery, College of Stomatology, West China University of Medical Sciences, Chengdu, Sichuan.
Leuk Res. 1992 Dec;16(12):1175-80. doi: 10.1016/0145-2126(92)90115-n.
After activation by interferon-gamma (INF-gamma) and lipopolysaccharide(LPS), mouse peritoneal macrophages were cocultured with P388 parental cell line (P388/PRT) and its adriamycin (ADM)-, cisplatin(CDDP)-, cyclophosphamide(CPM)-, and mitomycin-C(MMC)-resistant cell lines for one day at effector:target ratios (E:T) of 10:1, 5:1, and 2:1. The direct 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) cleavage assay and a new indirect MTT assay as well as clonogenic assay were used to quantitate activated macrophage-mediated cytotoxicity to these non-adherent leukemia targets. The results revealed that all the P388 cell lines can be suppressed efficiently by activated macrophages, but P388 CPM- and MMC-resistant cell lines (P388/CPM, P388/MMC) were more susceptible than P388/PRT while P388 ADM- and CDDP-resistant cell lines (P388/ADM, P388/CDDP) shared equal level of survival rates with P388/PRT. This study also showed that both non-activated and activated macrophages can produce formazan in a high level, which can interfere with the final results of direct MTT assay. The new indirect MTT assay can avoid such interference by separating the effectors from the targets before performing the MTT assay and reflects the real viability of the targets so the indirect MTT assay developed in this study could be a better way to examine cytostatic and cytotoxic effect of activated macrophages on non-adherent tumor cells in vitro.
经干扰素-γ（INF-γ）和脂多糖（LPS）激活后，将小鼠腹腔巨噬细胞与P388亲本细胞系（P388/PRT）及其对阿霉素（ADM）、顺铂（CDDP）、环磷酰胺（CPM）和丝裂霉素C（MMC）耐药的细胞系，以效应细胞与靶细胞比例（E:T）为10:1、5:1和2:1共培养一天。采用直接3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）裂解试验、一种新的间接MTT试验以及克隆形成试验，对活化巨噬细胞介导的对这些非贴壁白血病靶细胞的细胞毒性进行定量分析。结果显示，所有P388细胞系均可被活化巨噬细胞有效抑制，但P388 CPM耐药和MMC耐药细胞系（P388/CPM、P388/MMC）比P388/PRT更敏感，而P388 ADM耐药和CDDP耐药细胞系（P388/ADM、P388/CDDP）与P388/PRT的存活率水平相当。本研究还表明，未活化和活化的巨噬细胞均可高水平产生甲臜，这会干扰直接MTT试验的最终结果。新的间接MTT试验可通过在进行MTT试验前将效应细胞与靶细胞分离来避免这种干扰，并反映靶细胞的真实活力，因此本研究开发的间接MTT试验可能是体外检测活化巨噬细胞对非贴壁肿瘤细胞的细胞抑制和细胞毒性作用的更好方法。
