Abstract
A 51Cr-release test was developed for the detection of cell-mediated cytotoxicity against transplanted solid chemically induced rat tumours, and the findings were compared with those obtained in parallel tests using a microcytotoxicity assay. It was necessary to incorporate several modifications of the original Brunner assay (Brunner et al., 1968) in order to increase the sensitivity of the test as applied to long-term tumour lines maintained as glass-adherent cultures. These included: 1. preincubation of labelled tumour cells at 37 degrees C for 3-4 h before addition of effector cells; 2. preincubation of effector cells at 37 degrees C for 12 h before addition to target cells; 3. upon admixture of target and effector cells, intimate cell contact was established by gentle centrifugation; 4. after incubation of target and effector cells at 37 degrees C, a further incubation for 1 h at 45 degrees C was included to complete the release of 51Cr from non-viable cells. Maximal cytotoxicity of tumour-bearer effector cells was detected after 12 h incubation of lymphoid and target cells at a ratio of 200:1. Spleen cells from tumour-bearing rats during the first 2 weeks of tumour growth exhibited the same pattern of reactivity in the 51Cr-release test and the microcytotoxicity assay, although significant reduction in target-cell numbers was more frequently recorded using the microcytotoxicity assay. Tumour-bearer spleen cells showed reactivity against the homologous tumour as well as against unrelated tumours. Using either assay, pre-exposure of effector cells to 3M KC1 extracts of tumour was found to inhibit their effect on tumour cells most frequently in tests in which the effector-cell donor, soluble extract and target cell were of the same tumour line.
Highlights
Summary.-A 51Cr-release test was developed for the detection of cell-mediated cytotoxicity against transplanted solid chemically induced rat tumours, and the findings were compared with those obtained in parallel tests using a microcytotoxicity assay
The present study describes an attempt were washed twice with Hanks' balanced salt to modify the Brunner 5'Cr-release test (Brunner et al, 1968) in order to allow evaluation of tumour-bearer lymphoidcell-mediated cytotoxicity to cells from transplanted solid chemically induced rat tumours, cultured as long-term, glasssolution and suspended in Eagle'sHEPES buffer + 10% calf serum
The cells were sedimented by centrifugation, the supernatant was discarded, and the cells were resuspended in Eagle's-HEPES buffer prior to their use in the 51Cr-release test or microcytotoxicity assay
Summary
At 800 g for 5 min, 1 ml of supernatant and Target tumour cells and lymphoid cells.- 1 ml of remaining supernatant plus cell. The 51Cr release from tumour cells in the presence of normal lymphoid cells compared with medium control samples was determined in each experiment. Tests with tumour-bearer lymphoid cells which gave a % 51Cr release greater than the mean % 51Cr release plus two standard deviations for cells from that tumour line in the presence of normal lymphoid cells, were taken to represent a significant cytotoxic reaction (Table I). TABLE J.-51Cr Release from Tumour Cells in the Presence of Lymphoid Cells from Normal Rats
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