A long‐term ⁵¹chromium assay for in vitro cell‐mediated tumor immunity. Correlation with simultaneously performed microplate assays

Abstract

In vitro assays of cell-mediated tumor immunity utilizing 51Chromium (51Cr) labelling of cultured adherent solid tumor cells were designed which allowed an effector cell/target cell incubation time of 48 h without overriding spontaneous 51Cr release. In a series of 16 consecutive experiments, blood lymphocytes from healthy human donors, from patients with tumors unrelated to the cultural tumor target cells, and from colon carcinoma and melanoma patients were tested for their cytotoxic effects on various target cell pairs, human colon carcinoma, melanoma, or skin fibroblasts. The same reagents were used in simultaneously performed microplate and 51Cr assays. Results obtained by visual counting of microplate tests and by 24-h assays of 51Cr release or 51Cr retention correlated in 20/25 effector-cell/target-cell combinations. In a series of six consecutive experiments, lymph-node cells from untreated Wistar/Furth rats, and rats bearing either chemically-induced colon carcinoma NG-W1 or polyoma virus-induced sarcoma P-W13 were tested for their cytotoxicity on syngeneic rat colon carcinoma and sarcoma target cells. Criss-cross type experiments were performed by microplate and 15Cr techniques done in parallel. Results obtained by visual counting of microplate tests and by 48 h assays of 51Cr release or 51Cr retention correlated in 15/18 effector-cell/target-cell combinations.

In vitro cell‐mediated immune reactions of melanoma and colorectal carcinoma patients demonstrated by long‐term 51chromium assays

“…In analyzing differences in BL effects, an alternative to comparisons between BL populations would seem to be comparison of individual BL populations to test ampoules containing target cells exposed to media only. Media controls were performed in each assay, and as reported earlier (Steele et al, 1975) levels of 51Cr released and W r retained in these control ampoules were quite stable from test to test for a given target cell, averaging approximately 30-35 % spontaneous 51Cr release after 24 h incubation. However, the relationship between levels of 51Cr released and/or 51Cr retained in media controls and the levels of 51Cr released and/or 51Cr retained in the lowest effector-cell :target-cell dose of the least reactive BL population varied from test to test on the same target cells, and in each test from target cell to target cell.…”

“…Details of the 51Cr assay technique have been published (Steele et al, 1975). Human target cells were incubated, 4,000 cells/0.2 ml Waymouth's medium containing 20% fetal calf serum, in flatbottomed sterile 2 ml glass ampules (Flow Laboratories, Solna, Sweden) for 6-8 h at 37" C in a 5 % CO, atmosphere.…”

“…A 51chromium ( T r ) assay has recently been developed which allows long-term (24-48 h) assay of in vitro BL and lymph-node-cell (LNC) cytotoxicity to adherent solid tumor and fibroblast target cells without overriding spontaneous 51Cr release (Steele et al, 1975). In a previous report, LNC from rats bearing chemically induced colon carcinomas or polyoma virus-induced sarcomas demonstrated specific tumor-associated cytotoxicity when monitored by either 51Cr or microcytotoxicity assays done in parallel.…”

Natural Cytotoxicity against Chemically Induced Neurogenic Rat Tumors

“…The larger the radiation dose the greater the decrease in the population of cy cling cells. (3) The low percentage of back ground release at 24 h was independent of the amount of 5lCr uptake, and is usable for a 24-hour incubation assay similar to that of Steele et al [33].…”