A Rapid Method for the Separation of Functional Lymphoid Cell Populations of Human and Animal Origin on PVP‐Silica (Percoll) Density Gradients

Abstract

Subclasses of lymphocytes can be separated on gradients of non-toxic polyvinylpyrrolidone-coated colloidal silica (Percoll) by virtue of differential densities. Such gradients can yield functionally active lymphocyte populations after brief centrifugation. Gradients can be generated in a discontinuous step fashion and centrifuged in standard table-top laboratory centrifuges or as self-generating gradients during ultracentrifugation. The density medium has low viscosity and can be made isotonic for virtually any use. Gradients have proved useful in both human and experimental animal studies, and high percentage yields allow for separations from small cell numbers. Methods are described for separation of whole blood and lymphoid subpopuctions. The cytoxic capability of various density fractions was evaluated for mixed lymphocyte culture-induced allogeneic killing and spontaneous, so-called "natural" killer cell activity. The lower density associated with blast transformation allows for significant enrichments of stimulated cells from in vitro cultures. Higher thymidine incorporation, restimualtion in mixed lymphocyte reactions, and greater cytotoxic capacity are associated with these "blast" fractions.

Measles Virus Fusion Protein Presented in an Immune-stimulating Complex (Iscom) Induces Haemolysis-inhibiting and Fusion-inhibiting Antibodies, Virus-specific T Cells and Protection in Mice

“…Cells were cultured in flat-bottomed 24-well plates at a density of 1.5 × 106 cells per well in Iscove's modified Dulbecco's medium, supplemented with 2 mM-L-glutamine, penicillin (100 units/ml), streptomycin (100 p.g/ml), 5 × 10 -5 M-2-mercaptoethanol (IMDM-S), 10~ foetal calf serum and 5Yo interleukin-2 (IL-2)-containing supernatant from rat splenocytes, which had been stimulated for 24 h with concanavalin A, and in the presence of 0.5 ~tg/well or 20 ~tg/well u.v.-inactivated MV. After four days the cells were collected and lymphoblastoid cells were isolated by gradient centrifugation on PercoU (Kurnick et al, 1979). T cell clones were obtained from blastoid ceils, culturing the cells by limiting dilution in IMDM-S supplemented with 10K foetal calf serum and 5 ~ IL-2 supernatant and in the presence of 0.75 ~tg u.v.-inactivated MV and 3 x 105 irradiated (1500 rad) syngeneic spleen cells per well.…”

Human endothelial cell-lymphocyte interaction. Endothelial cells function as accessory cells necessary for mitogen-induced human T lymphocyte activation in vitro.

“…Somewhat surprisingly, EC that had been cultured in vitro for as long as 7 wk and transferred five times before use were also able to function as accessory cells. This was unexpected because Hirschberg et al (8) could no longer detect HLA-DR antigens with a complement-mediated microcytotoxicity assay on ECuv that had been cultured in vitro for 14 …”

Percoll density gradient separation of cells from human malignant effusions

“…Percoll has increasingly been used for separation of haematopoietic colony forming cells (CFCs) and lymphocyte subsets (Pertoft, 1970, Kurnick & Ostberg, 1979Olofsson et al, 1980). More recently, malignant cells derived from experimental tumours have been separated from nonneoplastic cells by centrifugation on Percoll (Bosslet et al, 1981, Hamburger et al, 1983b.…”