Limits...
xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

Martinez-Serra J, Gutierrez A, Muñoz-Capó S, Navarro-Palou M, Ros T, Amat JC, Lopez B, Marcus TF, Fueyo L, Suquia AG, Gines J, Rubio F, Ramos R, Besalduch J - Onco Targets Ther (2014)

Bottom Line: With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored.In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, University Hospital Son Espases, Palma de Mallorca, Balearic Islands, Spain.

ABSTRACT
The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

No MeSH data available.


Related in: MedlinePlus

Increases in the number of cells seeded into the wells, correlated with increases in CI.Notes: (A) We seeded different numbers of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence (F+) or absence (F−) of fibronectin. We observed in the RTCA graph the correlation between the B-CI (mean ± SEM from at least two [up to four] independent wells [2 hours]) and the number of cells seeded (cell-attaching stage). We highlight that in the absence of fibronectin, the values of B-CI were next to zero. (B) B-CI versus MTT assay. After 24 hours, we showed that the MTT (mean ± SEM) values correlated with those obtained in the RTCA (mean ± SEM) only in the presence of fibronectin (Spearman’s rho; P=0.046). MTT values in the presence or absence of fibronectin were similar. (C) The anti-VLA-4 antibody inhibits Jurkat cells from attaching to fibronectin. Mean CI value (mean ± SEM) for 50,000 cells/well, incubated in the presence of different concentrations of anti-VLA-4.Abbreviations: B-CI, baseline cell index; CI, cell index; F, fibronectin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RTCA, real-time cell analysis; SEM, standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061162&req=5

f2-ott-7-985: Increases in the number of cells seeded into the wells, correlated with increases in CI.Notes: (A) We seeded different numbers of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence (F+) or absence (F−) of fibronectin. We observed in the RTCA graph the correlation between the B-CI (mean ± SEM from at least two [up to four] independent wells [2 hours]) and the number of cells seeded (cell-attaching stage). We highlight that in the absence of fibronectin, the values of B-CI were next to zero. (B) B-CI versus MTT assay. After 24 hours, we showed that the MTT (mean ± SEM) values correlated with those obtained in the RTCA (mean ± SEM) only in the presence of fibronectin (Spearman’s rho; P=0.046). MTT values in the presence or absence of fibronectin were similar. (C) The anti-VLA-4 antibody inhibits Jurkat cells from attaching to fibronectin. Mean CI value (mean ± SEM) for 50,000 cells/well, incubated in the presence of different concentrations of anti-VLA-4.Abbreviations: B-CI, baseline cell index; CI, cell index; F, fibronectin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RTCA, real-time cell analysis; SEM, standard error of the mean.

Mentions: We seeded different amounts of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence or absence of fibronectin (Figure 2A). As expected, we observed that the B-CI values, as calculated by xCELLigence, increased when a higher amount of cells were seeded (Figure 2). Therefore, the greater the number of cells deposited on the bottom of the well, the greater the B-CI reading. We highlight that in the absence of fibronectin, the B-CI values were next to 0. Moreover, in order to compare our results with a conventional method used for cell viability measurements, we carried out an MTT assay on the same plate, after 24 hours. We show that the MTT/OD (490 nm) values correlated with those obtained in the RTCA (B-CI; P=0.046; Figure 2B). We also compared the values of the MTT assay when cells were incubated in the presence (F+) or absence (F−) of fibronectin. We could not detect significant differences between these two groups. This data suggests that fibronectin, under these experimental conditions, may not affect the viability/cell growth of the Jurkat cells. Finally, as adhesion to fibronectin occurs mainly through the VLA-4 integrin receptor, we incubated 50,000 Jurkat cells for 1 hour in the presence of different concentrations (25, 50, and 100 μg/mL) of an anti-VLA-4 antibody. The higher concentration of anti-VLA-4 corresponded with the higher inhibition of cell attachment to the bottom of the wells. A concentration of 100 μg/mL of anti-VLA-4 inhibited up to 60% of the CI (20 hours; 0.1704±0.0137) reached when cells were incubated only in the presence of fibronectin (20 hours; 0.4004±0.0292; Figure 2C). This data highlights the specificity of this integrin on the adherence of the leukemia cells on the RTCA plates.


xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

Martinez-Serra J, Gutierrez A, Muñoz-Capó S, Navarro-Palou M, Ros T, Amat JC, Lopez B, Marcus TF, Fueyo L, Suquia AG, Gines J, Rubio F, Ramos R, Besalduch J - Onco Targets Ther (2014)

Increases in the number of cells seeded into the wells, correlated with increases in CI.Notes: (A) We seeded different numbers of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence (F+) or absence (F−) of fibronectin. We observed in the RTCA graph the correlation between the B-CI (mean ± SEM from at least two [up to four] independent wells [2 hours]) and the number of cells seeded (cell-attaching stage). We highlight that in the absence of fibronectin, the values of B-CI were next to zero. (B) B-CI versus MTT assay. After 24 hours, we showed that the MTT (mean ± SEM) values correlated with those obtained in the RTCA (mean ± SEM) only in the presence of fibronectin (Spearman’s rho; P=0.046). MTT values in the presence or absence of fibronectin were similar. (C) The anti-VLA-4 antibody inhibits Jurkat cells from attaching to fibronectin. Mean CI value (mean ± SEM) for 50,000 cells/well, incubated in the presence of different concentrations of anti-VLA-4.Abbreviations: B-CI, baseline cell index; CI, cell index; F, fibronectin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RTCA, real-time cell analysis; SEM, standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061162&req=5

f2-ott-7-985: Increases in the number of cells seeded into the wells, correlated with increases in CI.Notes: (A) We seeded different numbers of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence (F+) or absence (F−) of fibronectin. We observed in the RTCA graph the correlation between the B-CI (mean ± SEM from at least two [up to four] independent wells [2 hours]) and the number of cells seeded (cell-attaching stage). We highlight that in the absence of fibronectin, the values of B-CI were next to zero. (B) B-CI versus MTT assay. After 24 hours, we showed that the MTT (mean ± SEM) values correlated with those obtained in the RTCA (mean ± SEM) only in the presence of fibronectin (Spearman’s rho; P=0.046). MTT values in the presence or absence of fibronectin were similar. (C) The anti-VLA-4 antibody inhibits Jurkat cells from attaching to fibronectin. Mean CI value (mean ± SEM) for 50,000 cells/well, incubated in the presence of different concentrations of anti-VLA-4.Abbreviations: B-CI, baseline cell index; CI, cell index; F, fibronectin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RTCA, real-time cell analysis; SEM, standard error of the mean.
Mentions: We seeded different amounts of Jurkat cells (40,000, 80,000, 120,000, and 240,000 cells/well) in the presence or absence of fibronectin (Figure 2A). As expected, we observed that the B-CI values, as calculated by xCELLigence, increased when a higher amount of cells were seeded (Figure 2). Therefore, the greater the number of cells deposited on the bottom of the well, the greater the B-CI reading. We highlight that in the absence of fibronectin, the B-CI values were next to 0. Moreover, in order to compare our results with a conventional method used for cell viability measurements, we carried out an MTT assay on the same plate, after 24 hours. We show that the MTT/OD (490 nm) values correlated with those obtained in the RTCA (B-CI; P=0.046; Figure 2B). We also compared the values of the MTT assay when cells were incubated in the presence (F+) or absence (F−) of fibronectin. We could not detect significant differences between these two groups. This data suggests that fibronectin, under these experimental conditions, may not affect the viability/cell growth of the Jurkat cells. Finally, as adhesion to fibronectin occurs mainly through the VLA-4 integrin receptor, we incubated 50,000 Jurkat cells for 1 hour in the presence of different concentrations (25, 50, and 100 μg/mL) of an anti-VLA-4 antibody. The higher concentration of anti-VLA-4 corresponded with the higher inhibition of cell attachment to the bottom of the wells. A concentration of 100 μg/mL of anti-VLA-4 inhibited up to 60% of the CI (20 hours; 0.1704±0.0137) reached when cells were incubated only in the presence of fibronectin (20 hours; 0.4004±0.0292; Figure 2C). This data highlights the specificity of this integrin on the adherence of the leukemia cells on the RTCA plates.

Bottom Line: With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored.In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, University Hospital Son Espases, Palma de Mallorca, Balearic Islands, Spain.

ABSTRACT
The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

No MeSH data available.


Related in: MedlinePlus