Abstract
A new assay method for natural killer (NK) cell activity was established using quantitative RT-PCR (RT-qPCR) to determine the gene expression of granzyme B (GzmB) and perforin 1 (Prf1). The RT-qPCR method was compared to a conventional cytotoxic assay. Upregulated expression of GzmB and Prf1 mRNA and enhanced cytotoxic activity were observed in splenocytes from lipopolysaccharide (LPS)-treated rats. A high correlation, R2 = 0.71, was observed between the gene expression of GzmB and the cytotoxic activity of splenocytes from these rats, indicating that GzmB RT-qPCR is a reliable alternative method to assess NK cell activity/activation. Remarkably, 12.6- to 59.7-fold upregulation of GzmB mRNA expression was observed in leukocytes, the spleen, and splenocytes from LPS-injected rats. Its upregulation appeared to be dose-dependent on the LPS concentration in the range of 0.01 - 0.1 mg/kg. Whereas, only 1.3- to 1.9-fold increase of cytotoxic activity was detected in splenocytes from the rats treated with LPS in the same range. From these, it is evident that, to assess NK cell activity/activation, the GzmB RT-qPCR method is highly sensitive compared with the conventional cytological assay. Furthermore, this GzmB RT-qPCR method is advantageous, as it does not require freshly prepared splenocytes and cell culture procedures. The convenience of GzmB RT-qPCR enables the use of whole blood, leukocytes, the spleen, and/or their frozen samples to evaluate NK cell activity/activation.
Highlights
natural killer (NK) cells participate in eliminating tumor cells and virus-infected cells through their cytotoxic activity
These results indicate that using RT-qPCR to measure granzyme B (GzmB) mRNA is a highly sensitive method to assess the activity of NK cells compared with conventional cytological assay using WST-1
These results suggest that RT-qPCR of GzmB mRNA is an alternative method to be applied to splenocytes and spleen, and leukocytes and whole blood
Summary
NK cells participate in eliminating tumor cells and virus-infected cells through their cytotoxic activity. Cytotoxic lactate dehydrogenase and WST-1/ WST-8 cell proliferation assays have been employed to measure NK activity [4] [5]. These assays do not require the labeling of target cells, and commercially available kits can be used. They still require time-consuming cytological procedures to prepare fresh splenocytes as effector cells and their co-culture with target cells. Another simple and easy method for measuring NK cell activity is required
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