Comparison of Several Methods for Isolation of Rat Peritoneal Mast Cells.

Abstract

Interest in the role of mast cells in various pathological and physiological phenomena has prompted the development of methods for isolating these cells. Mast cells have been isolated from rat peritoneal fluid (they constitute 2 4 % of the peritoneal cell population) by density gradient centrifugation through concentrated solutions of sucrose( 1 ) , Ficoll ( a high molecular weight polysaccharide) ( 2 ) or albumin(3). Although it has been claimed (2,3) that sucrose is inferior to albumin or Ficoll for isolating mast cells, there has been no detailed comparison of these 3 media. The experiments reported here were designed to provide a systematic comparison of sucrose, Ficoll and albumin solutions as isolation media for rat peritoneal mast cells.Methods. Mixed peritoneal cells were harvested from young male rats (250-300 g) of the Wistar strain according to the method of Uvnas and Thon ( 2 ) . The cells from 3 or 4 rats were pooled for each experiment. The animals were anesthetized with ether and exsanguinated. A section of midline abdominal skin was excised, a small hole was cauterized in the abdominal wall through which a buffered isotonic salt solution was introduced (see below). The hole was closed and the abdomen massaged gently for 90 seconds. The fluid, containing several types of cells, was then aspirated through a midline incision and centrifuged at 200 X g for 5 minutes at room temperature. T h e supernatant was discarded, and the cells were resuspended in 16-18 ml of buffered salt solution. Aliquots of mixed cell suspension were taken for mast cell counts and for determination of histamine. Eight ml of cell suspension were layered over the dense medium in a centrifuge tube. Centrifugation separated the cells into two layers, *Supported by grant H E 029.i3 from Nat. Heart Inst. Nat. Inst. Health. Publication 823 of the Division of Basic Health Sciences, Emory University.t Predoctoral trainee of USPHS Graduate Pharmacology Training Granlt 2T1 GM 179. one (mixed cells) at the interface of the light and heavy media and the other (mast cells) a t the bottom of the tube. T h e upper layer of fluid was removed with a pipette, and the interfacial layer of cells was similarly removed and resuspended in fresh buffered solution. The cells of this layer were washed, and samples were taken for histamine assay and mast cell counts. The sides of the centrifuge tube were wiped gently at the level of the interface to remove adhering cells, The dense medium containing mast cells was diluted with 2 ml of fresh, buffered salt solution and centrifuged (200 X g for 5 min). The mast cells were washed twice in fresh, buffered salt solution and finally resuspended in 8-9 ml. Aliquots were taken for estimation of histamine release as described below. All wash fractions and the media removed from the cells were acidified and stored frozen until assayed for histamine. Media.A buffered salt solution (pH 6.8-7.0) containing 150 mM NaC1, 2.7 mM KCI, 0.9 mM CaC12, 3 mM N a 2 H P 0 4 * 2H20, 3.5 mM KH2POl, 5.6 mM dextrose and 0.1% human...

Sodium Fluoride – a Stimulus for a Calcium-Triggered Secretory Process

“…proteins (antigen [21], cationic proteins derived from neutro phils [29,30] or basic protein from cobra venom [25]), polysaccharides (dextran [3]), adenosine derivatives (ATP [5]), polymer amines (compound 48/80 [34], ionophoreous antibiotics (A23187 [17]), and com plement factors (C3a and C5a [22]). All these agents seem to depend on calcium, de rived either from intracellular or extracellu lar sources as well as on metabolic energy to trigger an exocytotic secretory process.…”

Histamine-Releasing Effect of a Corticotrophin Derivative

“…Norn (17) and Johnson and Moran (12) have shown that, in the first place, only the mast cells present in the suspension constitute a source of histamine and, secondly, that fractionation of the suspension can diminish the sensitivity of the cells to the action of liberating agents.…”

Inhibition by Nicotinamide of an Homologous PCA Reaction and Antigen-Induced Histamine Release from Rat Peritoneal Mast Cells

“…After gentle massage of the abdomen for 90 sec, a suspension of peritoneal cells was aspirated through a midline incision. Mast cells were isolated by differen tial centrifugation through 37% solution of bovine serum albumin (fraction V, from Biomed, Poland), according to the method of Johnson and M oran [12], or in some experiments only washed 3 times by centrifugation (5 min) at 200 g. The mast cells were counted after staining with toluidine blue [15,20] and distributed into several tubes, so that each tube contained an approximately equal number of mast cells. The samples were incubated for 10 min at 37 °C in a bath shaker, challenged with antigen (final concentration 100 «g/ml) and incubated once more for 6 min at 37 °C.…”