Rs by centrifugation in the presence of LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway), and have been cultured as described above together with the addition of ten g/ml of phytohemagglutinin (SigmaAldrich, Milan, Italy). A single dose of CF (final concentration 5 l/ml) was administered to leukemia cells or lymphocytes; cells were collected following 24, 48, and 72 h of CF administration. Untreated cells served as controls. Trypan blue cell counting was performed at every experimental time point to evaluate the viable cell number.Cell viability assayCell proliferation and viability were analyzed at 450 nm by the WST-1 reagent (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) (Roche Diagnostics GmbH, Mannheim, Germany). The assay was according to the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Briefly, leukemia cells had been incubated in 96-well plates inside the presence or absence of CF (5 l/ml); right after 24, 48, and 72 h of incubation, WST-1 was added to each and every well, and cells have been additional incubated at 37 as much as two h. Colour improvement was monitored at 450 nm within a multiwell plate reader (Thermo Fisher Scientific, Shangai).Caspase-3 activity evaluationCaspase-3 activity was determined in leukemia cells using a colorimetric kit from Biovision (Milpitas, CA, USA) in accordance using the manufacturer’s guidelines.1445951-89-2 Data Sheet The assay is according to the spectrophotometric detection at 405 nm in the chromophore p-nitroaniline (pNA) just after cleavage from the labeled substrate DEVDpNA by caspase-3. Protein concentration in the cytosolic extracts was measured utilizing the Bradford strategy [24].Catalani et al. Journal of Experimental Clinical Cancer Research 2013, 32:63 http://jeccr/content/32/1/Page three ofDNA fragmentation analysisLDH activity and lactate release measurementThe genomic DNA fragmentation was evaluated by agarose gel electrophoresis of DNA isolates obtained by the salting-out method [25]. For this purpose, leukemia cells had been grown inside the presence or absence of CF 5 l/ml as much as 72 h; a optimistic control (cells treated for 6 h with 25 M etoposide) was also integrated.Fmoc-D-beta-indanylglycine In stock Soon after counting and washing, cells have been subjected to DNA extraction. The DNA samples have been very carefully resuspended in TE buffer; the nucleic acid concentration and purity were measured making use of a NanoDrop?ND-1000 spectrophotometer (Thermo-Scientific, Wilminton, DE, USA). 2 g of each and every sample was loaded onto 1.five TAE agarose gel; DNA laddering was visualized on a UV transilluminator by ethidium bromide staining. Photos have been obtained employing a Gel Doc 2000 (Bio-Rad Laboratories S.r.PMID:24293312 l, Segrate, MI, Italy).HIF-1 measurementHIF-1 quantification was performed in leukemia cells applying an enzyme-linked immunosorbent assay kit from Abcam (Cambridge, UK), in accordance with the manufacturer’s instructions. Colour improvement was evaluated at 450 nm in a multiwell plate reader (Thermo Fisher Scientific, Shangai). Protein concentration in cell extracts was measured utilizing the Bradford method [24].Western blot assay of GLUT-After 72 h of incubation within the presence or absence of CF (5 l/ml), leukemia cells had been centrifuged at 450 g for 10 min at area temperature; supernatants were collected to evaluate lactate release in the culture media when cell pellets have been employed for LDH activity determination. Lactate measurement was performed by means of an enzymatic assay in a hydrazine/glycine buffer (pH 9.two), containing 2 mg/ml -NAD+ and 16 units/ml LDH [26]. The absorbance on account of NADH fo.