Jost LM, Kirkwood JM & Whiteside TL. Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells. J Immunol Meth147: 153-165

Department of Medicine, University of Pittsburgh School of Medicine, PA 15213-2592.
Journal of Immunological Methods (Impact Factor: 1.82). 04/1992; 147(2):153-65. DOI: 10.1016/S0022-1759(12)80003-2
Source: PubMed


A tetrazolium compound, XTT, bioreducible to a water-soluble formazan was used to develop a simplified cellular cytotoxicity assay. Most (13/15 melanoma and 2/3 colon carcinoma cell lines tested metabolized XTT greater than 50 times more efficiently than the lymphoid effector cells, and thus the test could be performed without separation of the effector from the target cells. The XTT assay (XTT-A) was compared to the standard 51chromium-release assay (51CrA) in terms of sensitivity as well as intra- and interassay variability using low effector to target cell (E:T) ratios and both short and long incubation periods. The correlation coefficient (r) for percent specific lysis (%SL: 35.0 +/- 15.0 versus 30.2 +/- 15.8) or lytic units (LU20/10(7) effector cells: 405 +/- 208 versus 357 +/- 227) between XTT-A and 51CrA was 0.86 for 4 h XTT-A and 51CrA (n = 37). Due to a poor performance of the 51CrA after 24 h incubation of effector and target cells, the correlation coefficient for 24 h assays was reduced to 0.79 (n = 44,%SL = 63.3 +/- 23.9 versus 55.5 +/- 26.6, and LU = 1267 +/- 982 versus 1017 +/- 691). Inter- and intra-assay variability of XTT-A were significantly lower than those for 51CrA. The total background values for XTT-A and 51CrA were similar in 4 h cytotoxicity assay and lower for XTT-A in assays with 24 h incubation. The sensitivity, in terms of discrimination between effector cells with different lytic capacity and targets with different susceptibility, was identical. The XTT-A was simpler, cheaper, and safer to perform than the 51CrA. Furthermore, the XTT-A was suitable for long-term assays and allowed experiments without requiring trypsinization of tumor cells grown in 96-well plates prior to testing.

12 Reads
  • Source
    • "MCF7 cells express hypoglycosylated mucin (13). This cell line was used as the target cell line in an XTT® assay (Roche Diagnostics Corp., Indianapolis, IN, USA) (14,15) performed according to the manufacturer’s instructions. The TGFα-PE38 or effector cells were tested at a concentration of effector-to-target cell ratio of 1.25. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the present study was to determine whether the combination of two modalities of immunotherapy, targeting two different tumor antigens, may be feasible and non-toxic, yet enhance the killing of a human breast cancer cell line. The first modality was tumor growth factor α-Pseudomonas exotoxin 38 (TGFα-PE38), which specifically targets and kills tumor cells that express the epidermal growth factor receptor. The second modality was mucin-1 (MUC1)-specific cytotoxic T lymphocytes (CTLs), generated by MUC1 stimulation of peripheral blood mononuclear cells, to target the human breast cancer cell line, MCF7. TGFα-PE38 exhibited specific lysis of the MCF7 cells in a concentration- and time-dependent manner. TGFα-PE38 did not kill the normal hematopoietic stem cells or CTLs. Furthermore, TGFα-PE38 was not inhibitory for the growth or differentiation of the normal human hematopoietic stem cells into erythroid and myeloid colonies. In addition, TGFα-PE38 did not inhibit the killing function of CTLs, either when preincubated or co-incubated with CTLs. Finally, therapeutic enhancement was observed, in that TGFα-PE38 and CTLs were additive in the specific lysis of the MCF7 cells. These two modalities of immunotherapy may be beneficial for humans with breast cancer with or without other therapies, including autologous hematopoietic stem cell transplantation, specifically for purging cancer cells from hematopoietic stem cells prior to transplantation.
    Oncology letters 06/2014; 7(6):2113-2117. DOI:10.3892/ol.2014.1969 · 1.55 Impact Factor
  • Source
    • "Here, we employed XTT and SRB assays to more convincingly examine effects of 8 ginsenosides on various cancer cells. SRB assay measures total biomass, namely number of cells, by staining cellular proteins with Sulforhodamine B, whereas dye reduction by mitochondrial reductants on cell surface is determined in XTT assay, actually measuring pyridine nucleotide redox status of live cells [14,17]. We used 5 human cancer cell lines; NCI-H23 lung cancer cells, PC-3 prostate cancer cells, ACHN and Caki-1 renal cancer cells, and K562 leukemia cells, since ginsenosides might have cell type- or tissue-specific cytotoxic effects. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms. Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model. PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-beta-cyclodextrin (MbetaCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo. This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies.
    BMC Complementary and Alternative Medicine 07/2013; 13(1):194. DOI:10.1186/1472-6882-13-194 · 2.02 Impact Factor
  • Source
    • "Cell viability was determined by the XTT test as described previously [23]. Briefly, cells were treated for 48 h, the old culture medium was replaced with a fresh one containing XTT (100 μg/ml) and PMS (1.0 μg/ml), and incubated for 4 h. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract. Betulinic acid was isolated from methanolic extract by crystallization and chromatography techniques. S.C. methanolic extract was analyzed by UV-Vis spectrophotometry, FTIR, LC-MS, and HPLC. Antiangiogenic effect was studied on rat aortic rings, matrigel tube formation, cell proliferation and migration, and expression of vascular endothelial growth factor (VEGF). Antitumor effect was studied using a subcutaneous tumor model of HCT 116 colorectal carcinoma cells established in nude mice. Analysis by HPLC, LC-MS and FTIR confirm presence of betulinic acid in S.C. methanolic extract. Quantitative analysis by HPLC indicates presence of betulinic acid in S.C. extract at 5.42 +/- 0.09% (w/w). Antiangiogenesis study showed potent inhibition of microvessels outgrowth in rat aortic rings, and studies on normal and cancer cells did not show any significant cytotoxic effect. Antiangiogenic effect was further confirmed by inhibition of tube formation on matrigel matrix that involves human endothelial cells (IC50 = 17.6 +/- 2.9 mug/ml). S.C. extract also inhibited migration of endothelial cells and suppressed expression of VEGF. In vivo antiangiogenic study showed inhibition of new blood vessels in chicken embryo chorioallantoic membrane (CAM), and in vivo antitumor study showed significant inhibition of tumor growth due to reduction of intratumor blood vessels and induction of cell death. Collectively, our results indicate S. campanulatum as antiangiogenic and antitumor candidate, and a new source of betulinic acid.
    BMC Complementary and Alternative Medicine 07/2013; 13(1):168. DOI:10.1186/1472-6882-13-168 · 2.02 Impact Factor
Show more