Determination of Cell Viability

Hanks, John H.; Wallace, John H.

doi:10.3181/00379727-98-23985

Cited by 253 publications

(73 citation statements)

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“…This effect probably cannot be attributed to a pathological condition in the animals tested, since all sera of 43 "normal" rabbits were toxic to 60 to 100 per cent of autologous cells. It may be argued that the presumption of cell death by uptake of eosin dye (6,15) is unjustified. That is, the cells may have become merely more permeable to the dye upon pre-exposure to fresh serum.…”

Section: Discussionmentioning

confidence: 99%

Destruction of Epidermal Cells in Vitro by Autologous Serum From Normal Animals

Terasaki

Chamberlain

1962

The Journal of Experimental Medicine

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Although normal serum is sometimes said to be toxic to autologous cells in vitro (1), to our knowledge, data of such autotoxic effects have been previously reported only for spermatozoa (2, 3). Indeed, autologous serum is often used as the best nutrient medium for cells (4). Evidence will be presented here that fresh normal sera from rabbits and rats are lethal in vitro to autologous epidermal cells. In the test system employed, 70 to 100 per cent of epidermal cells were killed within 2 hours at 37°C. Materials and MethodsAnimals.--The following strains were used: Dutch and Zealand rabbits, Long Evans rats, C57 mice, and mongrel guinea pigs.Cell Preparation.--Epidermal cells from the basal or malpighian layer of the epidermis were prepared by the trypsinization method of Billingham and Reynolds (5) with a few modifications. A 0.010 to 0.015 inch split thickness of skin was obtained from the lateral surface of the body with a Brown electrodermatome. It was then scraped free of dead cuticular cells and connective tissue, and placed with its dermal surface down in a Petri dish containing 0.25 per cent trypsin (Difco 1:250) in versene-BSS (0.002 ~ tetrasodium ethylenediaminetetraacetate in Hanks's balanced salt solution). Upon incubation in a shaker at 37°C for 20 minutes, areas of the skin which appeared clear were excised and the remaining skin allowed to incubate for periods up to 1~ hours. The trypsinized pieces were then washed in saline and in versene-BSS, placed with the dermis of the skin up in a dry Petri dish, and blotted free of fluid. After peeling back the dermis the exposed surface of the epidermis was scraped with a scalpel. The freed malpighian cells were suspended in versene-BSS, pooled, centrifuged at 1000 g for 1 minute, and resuspended in BSS. Later tests have indicated that the use of versene could be eliminated completely.

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Section: Discussionmentioning

confidence: 99%

Destruction of Epidermal Cells in Vitro by Autologous Serum From Normal Animals

Terasaki

Chamberlain

1962

The Journal of Experimental Medicine

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“…Assay: At day 6 of culture, the cells from triplicate cultures were harvested, pooled, and washed twice. The viability of the cultured cells in the positive control (unfracfionated CBA spleen cells together with unfractionated mitomycin C-treated BALB/c spleen cells) was determined by the eosln dye exclusion method (28) and the cell concentration adjusted to a 5 X 10 ~ viable lymphocytes/ml. Usually a dilution of 1 to 4, 1 to 16, and 1 to 64 of these cells was assayed in FEM supplemented with 10% heat-inactivated FCS.…”

Section: Enumeration Of Antibody-forming Cells-cells Forming Antibodmentioning

confidence: 99%

Cell-Mediated Immune Response in Vitro

Wagner

Feldmann

Boyle

et al. 1972

The Journal of Experimental Medicine

151

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The combined use of in vitro culture techniques together with efficient cell separation methods have revealed that macrophages are essential participants in the initiation of some antibody responses (1-4). The experimental design used was to obtain "purified" (macrophage-depleted) lymphocytes by a cell separation procedure and to demonstrate that these cells could not respond to a particular antigen unless macrophages were also present. Several mechanisms of macrophage function during the induction of the humoral immune response have been proposed. Since it was found that in vitro responses to particulate antigens, such as whole erythrocytes, required the participation of macorphages, whereas responses to smaller sized antigens, such as "soluble sheep red blood cell antigen" (3), or polymeric flagellin of Salmonella adelaide (POL) 1 did not, it was proposed that the role of macrophages may therefore be merely to reduce antigenic particles to a smaller and more immunogenic size. Experiments demonstrating that macrophage supernatants enhance responses to erythrocytes (5, 6) are consistent with this simple mechanism of macrophage function. Recently it was found that the immune response to thymus-dependent antigens, even those of small size, such as monomeric flagellin (MON), dinitrophenylated MON (DNP MON), or DNP fowl gamma globulin (DNP F~G), all required the presence of macrophages, whereas responses to the same antigenic determinants (DNP) on polymeric carriers (i.e. POL or DNP POL) which are thymus independent were also found to be

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“…After clumps had been allowed to settle out over 5 min the cell suspension was decanted. Counts of viable nucleated cells, viability being assessed by eosin exclusion (Hanks and Wallace, 1950), were made using a hemocytometer.…”

Section: Cell Suspensionsmentioning

confidence: 99%

REGULATION OF THE IMMUNE RESPONSE BY IgM ANTIBODY: A PARADOXICAL SUPPRESSION OF THE IN VITRO PRIMARY IMMUNE RESPONSE TO SHEEP ERYTHROCYTES BY PASSIVE IgM.

Schrader

1973

Aust J Exp Biol Med

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Summary. Using a purified preparation of mouse anH-shwp er>'throcyte" IgM, a marked suppri?ssion of the in vitro primary respanse of mouse spleen cells to shi-ep rwl cells wa.s cIcmoiLStrated, This fffctt wa.s shown ti» be specific iu lliat a primary-rrsponse to the dinltr{)phenyl dt'temiinant wus unaffected. The response to donk''y erythrocytt'.s, however, wa.s siippre.sspe supprestiive both in vitro and tn vivo.

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Determination of Cell Viability

Cited by 253 publications

References 0 publications

Destruction of Epidermal Cells in Vitro by Autologous Serum From Normal Animals

Destruction of Epidermal Cells in Vitro by Autologous Serum From Normal Animals

Cell-Mediated Immune Response in Vitro

REGULATION OF THE IMMUNE RESPONSE BY IgM ANTIBODY: A PARADOXICAL SUPPRESSION OF THE IN VITRO PRIMARY IMMUNE RESPONSE TO SHEEP ERYTHROCYTES BY PASSIVE IgM.

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