Malaria pigment accelerates MTT – formazan exocytosis in human endothelial cells

Abstract

Haemozoin is a by-product of haemoglobin digestion by intraerythrocytic malaria parasites, which induces immunologic responses on different tissues, including endothelial cells. In the present paper, the incubation of human microvascular endothelial cells with haemozoin significantly inhibited MTT reduction, a measure of cytotoxicity, without increasing the release of cytoplasmic lactate dehydrogenase. Moreover, haemozoin did not induce apoptosis or cell cycle arrest nor decreased the number of live cells, suggesting that cells viability itself was not affected and that the inhibition of MTT reduction was only apparent and probably due to accelerated MTT-formazan exocytosis. After 30 min of MTT addition, a significant increase in the % of cells exocytosing MTT formazan crystals was observed in haemozoin-treated cells compared with control cells. Such an effect was partially reversed by the addition of genistein, an inhibitor of MTT-formazan exocytosis. The rapid release of CXCL-8, a preformed chemokine contained in Weibel–Palade bodies, confirmed that haemozoin induces a perturbation of the intracellular endothelial trafficking, including the exocytosis of MTT-formazan containing vesicles. The haem moiety of haemozoin is responsible for the observed effect. Moreover, this work underlines that MTT assay should not be used to measure cytotoxicity induced by haemozoin and other methods should be preferred.

Effects of Dendrobium Polysaccharides on the Functions of Human Skin Fibroblasts and Expression of Matrix Metalloproteinase-2 under High-Glucose Conditions

“…Conversely, dead cells do not exhibit this property. Dimethyl alum dissolves the blue-purple crystals in the cells, and the color depth is proportional to the amount of formazan [17,18].…”

Mulberrofuran G, a Mulberry Component, Prevents SARS-CoV-2 Infection by Blocking the Interaction between SARS-CoV-2 Spike Protein S1 Receptor-Binding Domain and Human Angiotensin-Converting Enzyme 2 Receptor

“…HEK293T cells were seeded into 96-well plates at 5 × 10 4 cells/well, and MG was added to the wells at concentrations of 0–50 μM. MTT solution was added to each well after 24 h, and the cells were incubated for a further 30 min [ 21 , 22 ]. Subsequently, 1 mL dimethyl sulfoxide was added before the absorbance at 540 nm was measured using an Epoch Microplate Reader (BioTek, Winooski, VT, USA).…”

Regulation of Dendrobium Polysaccharides on Proliferation and Oxidative Stress of Human Umbilical Vein Endothelial Cells in the High Glucose Environment

“…Set the temperature constantly of the water bath to 37°C for 40 min, add the color reagent and mix well, pour it into a 1 cm optical path cuvette after 10 min, adjust to zero with distilled water, and compare the color with a microplate reader at a wave length of 550 nm. According to the formula SOD activity ðU/mgprotÞ = ½ðcontrol tube − measurement tubeÞ/control tube ÷ 50% × ðtotal volume of reaction solution /sampling amountÞ ÷ protein content in tissue ðmgprot/mLÞ, to calculate SOD activity, no clear sample is added to the control tube, and the others are the same as the measurement tube [13][14][15].…”