| | Re: Quick Cell Counting Methods
Method on tube : dispense 0.5 ml cells suspension in 3 ml tube and centrifuge at 250 g for 10 min. at room temperature. Remove supernatant medium by vacuum aspiration and cells are resuspended and fixed in 200 Ál of 50% (v/v) ethanol for 30 min., centrifuge and followed by vacuum aspiration fixative must be removed and to make sure that the remaining alcohol is completely evaporated. Dispense 100 Ál of 0.2% (w/v) Janus Green B in PBS, resuspeding cells and incubate for 3 min. at room temperature. Centrifuge as above and remove stain using a vacuum aspirator, and add 2 ml of tap water or PBS (8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4•2H2O, 0.2 g/L KH2PO4, adjust to pH 7.2) and destained for 1 min. by gentle shaking, rinse 3 time, following centrifugation each time. Centrifuge as above and to draw off the supernatant and the stained cells are immediately lysed in 250 Ál of 0.5 N HCl. Mix accurately and to transfer 200 Ál of it in MTP and read the Abs at 595 nm using a microplate reader. Cell numbers were calculated from absorbance readings using the formula : Cell Number = (absorbance-0.00462)/0.00006926.