Nonspectific cytotoxic cells in fish ( [formula omitted][formula omitted]) VI. Flow cytometric analysis

Abstract

Nonspecific cytotoxic cells (NCC) from channel catfish ( Ictalurus punctatus ) were enriched using flow cytometry (EPICS R V, 753). Parameters of forward angle and 90° light scatter were utilized to sort cytolytically functional populations of NCC. Fullbright (GR. II) 9.75 μm diameter fluorescent polystyrene microspheres were used for optical alignment. Cells obtained from the anterior kidney, spleen and peripheral blood were analyzed by one and/or two parameters of forward angle light scatter (FALS) and log integral 90° light scatter (L90°LS). Populations identified from FALS or 90°LS histograms were sorted and cells were tested for cytotoxicity against NC-37 target cells. Anterior kidney contained at least four different sized populations of NCC. Spleen cells were sorted by FALS and four different populations of NCC detected. Peripheral blood contained little NCC activity. Enrichment of NCC activity from anterior kidney tissue was compared using Percoll R density gradient separation and flow cytometry. Three to four fold higher cytotoxic activity was obtained from sorted cells compared to the population obtained from 45.5% Percoll R. Additional experiments to determine the enrichment of sorted NCC were conducted by analyzing the number of enriched NCC required to kill an individual NC-37 target cell. Approximately 1.5 - 2.0 NCC could lyse a chromium-51 labelled target cell. These studies demonstrated that NCC could be enriched by size and granularity discrimination using flow cytometry. Effector:target cell ratios of 10:1 and lower produced significant lysis when sorted cells were analyzed.