Spontaneous histamine secretion during isolation of rat cardiac mast cells

Abstract

Enzymatic dispersion of cardiac mast cells affects their response to secretagogues.

A novel technique for isolating functional mast cells from the heart

“…Slides were prepared using 100 µL of the isolation sample, diluted with 900 µL of 4 % paraformaldehyde in PBS and spun onto a glass slide for 10 min at 600 rpm using a cyto-spin centrifuge (CytoTek, Sakura). To characterize the phenotype, the isolated cells were stained with alcian blue and counterstained with safranin (Sigma Chemical Co., St. Louis, MO) and compared to comparably stained cardiac mast cells obtained by enzymatic digestion as previously described [20]. Additional slides were blocked in 1.5 % BSA at room temperature for 30 min and incubated at 4 ºC overnight with primary antibodies directed against chymase (Mast Cell Protease 1, goat anti-rat polyclonal antibody) or tryptase (Mast Cell Tryptase rabbit anti-rat polyclonal antibody), both at a 1:100 dilution (Santa Cruz BioTech., CA).…”

“…Our laboratory has demonstrated that cardiac mast cells are responsive to specific secretagogues (i. e., compound 48/80 & endothelin) in the intact heart [1,3,4,18]. Conventional techniques for cardiac mast cell isolation involve enzymatic digestion of the myocardium using bacterial collagenase [19][20][21][22][23][24]. However, we recently demonstrated that collagenase isolation of cardiac mast cells causes cellular degradation resulting in spontaneous histamine release, yielding mast cells that are minimally responsive to secretagogues [20].…”

“…Conventional techniques for cardiac mast cell isolation involve enzymatic digestion of the myocardium using bacterial collagenase [19][20][21][22][23][24]. However, we recently demonstrated that collagenase isolation of cardiac mast cells causes cellular degradation resulting in spontaneous histamine release, yielding mast cells that are minimally responsive to secretagogues [20]. Therefore, a technique allowing for the isolation of cardiac mast cells which remain functionally responsive to secretagogues would provide a powerful tool to fully elucidate the role of mast cells in cardiac remodeling.…”

Rat cardiac mast cell maturation and differentiation following acute ventricular volume overload

“…Cardiac mast cells were isolated following mechanical and enzymatic dispersion of the whole rat heart as previously described [13]. The entire heart was utilized for mast cell isolation to ensure a suffi cient yield of cells for the assessment of maturation and differentiation.…”

“…We previously confi rmed that rat cardiac mast cells can be classifi ed as "Connective Tissue-type" mast cells by the affi nity of these cells for safranin staining [13]. Specifi c classifi cation of the extent of maturation and differentiation of "Connective Tissue-type" granules was previously determined to consist of four morphological stages (I to IV) corresponding to a progression from predominately alcian-blue staining to safranin staining in mast cells from the heart and peritoneum of the rat [8][9][10].…”