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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

Drug cytotoxicity monitoring of Jurkat/L1236 cells in the presence/absence of Fibronectin: RTCA vs MTT assay.Notes: (A) We incubated Jurkat/L1236 cells (treatment point [+]) in the presence of trabectedin (T) (10 nM) + FasL (50 ng/mL), bendamustine (200 μM), oxaliplatin (100 μM), trabectedin (10 nM) + FasL (50 ng/mL), or bendamustine + oxaliplatin (up to 24 hours). Fibronectin allowed robust monitoring of drug-induced cytotoxicity. Clear decreases of the B-CI correlated with the presence of a large number of apoptotic cells as observed by microscopy (data not shown). (B) RTCA vs MTT: Jurkat/L1236 cells were incubated in the presence of T (1, 10, and 100 nM), D (0.1, 1, and 10 μM), Cis (0.1, 1, and 10 μM), and G (1, 10, and 100 μM) (up to 24–48 hours). The presence vs absence of fibronectin did not appear to affect drug-induced toxicity for Jurkat/L1236 cells using the MTT method, suggesting that fibronectin does not protect Jurkat cells under these experimental conditions.Abbreviations: B-CI, baseline cell index; Cis, cisplatin; D, doxorubicin; FasL, Fas ligand; G, gemcitabine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RTCA, real-time cell analysis; vs, versus.
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f4-ott-7-985: Drug cytotoxicity monitoring of Jurkat/L1236 cells in the presence/absence of Fibronectin: RTCA vs MTT assay.Notes: (A) We incubated Jurkat/L1236 cells (treatment point [+]) in the presence of trabectedin (T) (10 nM) + FasL (50 ng/mL), bendamustine (200 μM), oxaliplatin (100 μM), trabectedin (10 nM) + FasL (50 ng/mL), or bendamustine + oxaliplatin (up to 24 hours). Fibronectin allowed robust monitoring of drug-induced cytotoxicity. Clear decreases of the B-CI correlated with the presence of a large number of apoptotic cells as observed by microscopy (data not shown). (B) RTCA vs MTT: Jurkat/L1236 cells were incubated in the presence of T (1, 10, and 100 nM), D (0.1, 1, and 10 μM), Cis (0.1, 1, and 10 μM), and G (1, 10, and 100 μM) (up to 24–48 hours). The presence vs absence of fibronectin did not appear to affect drug-induced toxicity for Jurkat/L1236 cells using the MTT method, suggesting that fibronectin does not protect Jurkat cells under these experimental conditions.Abbreviations: B-CI, baseline cell index; Cis, cisplatin; D, doxorubicin; FasL, Fas ligand; G, gemcitabine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RTCA, real-time cell analysis; vs, versus.

Mentions: In order to check if fibronectin allowed the real-time monitoring of the cytotoxic action of several compounds, we incubated Jurkat and L1236 cells with different drugs, such as FasL (50 ng/mL), oxaliplatin (100 μM), bendamustine (200 μM), or trabectedin (10 nM). As seen in Figure 4A, the cell growth and the cytotoxic response were only monitored in the presence of fibronectin. Furthermore, we incubated Jurkat and L1236 cell lines with several chemotherapeutic agents at different drug concentrations: trabectedin (1, 10, and 100 nM), doxorubicin (0.1, 1, and 10 μM), cisplatin (0.1, 1, 10 μM), and gemcitabine (1, 10, 100 μM) (up to 24–48 hours). Once finished the RTCA experiment, we performed an MTT measurement on the same plate. We include in Figure 4B the changes in the percentage values for each dose with respect to the control (non-treated), for both the RTCA and the MTT method. We simultaneously treated Jurkat/L1236 cell lines with the same drug concentrations in a 96-well plate in the absence of fibronectin. In Figure 4B, we show that the results between RTCA and MTT in the presence of fibronectin are comparable. Moreover, we also show that the MTT values are similar when cells are incubated in the absence/presence of fibronectin under these experimental conditions (Figure 4B).


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)

Drug cytotoxicity monitoring of Jurkat/L1236 cells in the presence/absence of Fibronectin: RTCA vs MTT assay.Notes: (A) We incubated Jurkat/L1236 cells (treatment point [+]) in the presence of trabectedin (T) (10 nM) + FasL (50 ng/mL), bendamustine (200 μM), oxaliplatin (100 μM), trabectedin (10 nM) + FasL (50 ng/mL), or bendamustine + oxaliplatin (up to 24 hours). Fibronectin allowed robust monitoring of drug-induced cytotoxicity. Clear decreases of the B-CI correlated with the presence of a large number of apoptotic cells as observed by microscopy (data not shown). (B) RTCA vs MTT: Jurkat/L1236 cells were incubated in the presence of T (1, 10, and 100 nM), D (0.1, 1, and 10 μM), Cis (0.1, 1, and 10 μM), and G (1, 10, and 100 μM) (up to 24–48 hours). The presence vs absence of fibronectin did not appear to affect drug-induced toxicity for Jurkat/L1236 cells using the MTT method, suggesting that fibronectin does not protect Jurkat cells under these experimental conditions.Abbreviations: B-CI, baseline cell index; Cis, cisplatin; D, doxorubicin; FasL, Fas ligand; G, gemcitabine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RTCA, real-time cell analysis; vs, versus.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ott-7-985: Drug cytotoxicity monitoring of Jurkat/L1236 cells in the presence/absence of Fibronectin: RTCA vs MTT assay.Notes: (A) We incubated Jurkat/L1236 cells (treatment point [+]) in the presence of trabectedin (T) (10 nM) + FasL (50 ng/mL), bendamustine (200 μM), oxaliplatin (100 μM), trabectedin (10 nM) + FasL (50 ng/mL), or bendamustine + oxaliplatin (up to 24 hours). Fibronectin allowed robust monitoring of drug-induced cytotoxicity. Clear decreases of the B-CI correlated with the presence of a large number of apoptotic cells as observed by microscopy (data not shown). (B) RTCA vs MTT: Jurkat/L1236 cells were incubated in the presence of T (1, 10, and 100 nM), D (0.1, 1, and 10 μM), Cis (0.1, 1, and 10 μM), and G (1, 10, and 100 μM) (up to 24–48 hours). The presence vs absence of fibronectin did not appear to affect drug-induced toxicity for Jurkat/L1236 cells using the MTT method, suggesting that fibronectin does not protect Jurkat cells under these experimental conditions.Abbreviations: B-CI, baseline cell index; Cis, cisplatin; D, doxorubicin; FasL, Fas ligand; G, gemcitabine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RTCA, real-time cell analysis; vs, versus.
Mentions: In order to check if fibronectin allowed the real-time monitoring of the cytotoxic action of several compounds, we incubated Jurkat and L1236 cells with different drugs, such as FasL (50 ng/mL), oxaliplatin (100 μM), bendamustine (200 μM), or trabectedin (10 nM). As seen in Figure 4A, the cell growth and the cytotoxic response were only monitored in the presence of fibronectin. Furthermore, we incubated Jurkat and L1236 cell lines with several chemotherapeutic agents at different drug concentrations: trabectedin (1, 10, and 100 nM), doxorubicin (0.1, 1, and 10 μM), cisplatin (0.1, 1, 10 μM), and gemcitabine (1, 10, 100 μM) (up to 24–48 hours). Once finished the RTCA experiment, we performed an MTT measurement on the same plate. We include in Figure 4B the changes in the percentage values for each dose with respect to the control (non-treated), for both the RTCA and the MTT method. We simultaneously treated Jurkat/L1236 cell lines with the same drug concentrations in a 96-well plate in the absence of fibronectin. In Figure 4B, we show that the results between RTCA and MTT in the presence of fibronectin are comparable. Moreover, we also show that the MTT values are similar when cells are incubated in the absence/presence of fibronectin under these experimental conditions (Figure 4B).

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