xcelligence sp real-time cell analyzer  (Agilent technologies)

Journal: International Journal of Molecular Sciences

Article Title: Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)

doi: 10.3390/ijms25168984

Figure Lengend Snippet: Pharmacological characterization of CCR2 constructs using radioligand binding and functional assays. ( A ) Schematic representation of the two CCR2 constructs designed for this study, CCR2-HiBiT, and CCR2-HaloTag-HiBiT, containing the two tags in the intracellular interface of the receptor. The representation also depicts the binding sites where the different compounds bind. ( B , C ) Homologous displacement curves of [ 3 H]CCR2-RA-[ R ] ( B ) or [ 3 H]INCB3344 ( C ) specific binding by increasing concentrations of non-labeled CCR2-RA-[ R ] or INCB3344, respectively, in membranes from U2OS cells stably expressing CCR2 (referred as CCR2 in legend), or from HEK293T cells transiently transfected with CCR2-HiBiT or CCR2-HaloTag-HiBiT (referred as CCR2-HiBiT and CCR2-HaloTag-HiBiT in legend). Radioligand binding data are shown as mean ± SEM of three independent experiments performed in duplicates. ( D ) Representative vehicle-corrected, normalized Cell Index (CI) traces measured in xCELLigence after stimulation of HEK293T cells transfected with CCR2-HiBiT with vehicle (PBS) or increasing concentrations of CCL2. Data are shown as representative mean CI values of a single experiment performed in duplicate. ( E ) Combined concentration-response curves of CCL2 on HEK293T cells transfected with CCR2-HiBiT or CCR2-HaloTag-HiBiT. CCL2 cellular response was derived from the vehicle-corrected, normalized CI traces and is expressed as the maximum peak response within the first 15–20 min after stimulation. Data are shown as mean ± SEM of at least three independent experiments performed in triplicates. Statistical differences between normalized responses at the highest CCL2 concentrations were analyzed using an unpaired t -test analysis: * p < 0.05.

Article Snippet: The xCELLigence Real-Time Cell Analyzer (RTCA) SP system (Agilent, Santa Clara, CA, USA) was used to measure CCL2-induced morphology changes of HEK293T cells transiently transfected with CCR2 constructs, using the impedance of electron flow as a readout.

Techniques: Construct, Binding Assay, Functional Assay, Labeling, Stable Transfection, Expressing, Transfection, Concentration Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)

doi: 10.3390/ijms25168984

Figure Lengend Snippet: xCELLigence assays to investigate functional inhibition of CCR2-HaloTag-HiBiT after inducing degradation. ( A ) Representative vehicle-corrected, normalized Cell Index (CI) xCELLigence traces obtained with transfected HEK293T cells pretreated with the indicated compounds at 1 µM for 3 h before stimulation with 60 nM CCL2. Representative data are shown as mean ± SD of a single experiment performed in triplicate. ( B ) CCL2 cellular response was derived from the vehicle-corrected, normalized CI traces and is expressed as the area under the curve (AUC) within the first 20 min after stimulation. AUC values were normalized to the response of 60 nM CCL2 on its own for comparison. Data are shown as mean ± SEM of at least three independent experiments performed in triplicates. Statistical differences between normalized CCL2 responses were analyzed using one-way ANOVA with Tukey’s post-hoc test: ns, not significant, * p < 0.05, *** p < 0.001.

Article Snippet: The xCELLigence Real-Time Cell Analyzer (RTCA) SP system (Agilent, Santa Clara, CA, USA) was used to measure CCL2-induced morphology changes of HEK293T cells transiently transfected with CCR2 constructs, using the impedance of electron flow as a readout.

Techniques: Functional Assay, Inhibition, Transfection, Derivative Assay, Comparison

Journal: Cancers

Article Title: SMAD7 Sustains XIAP Expression and Migration of Colorectal Carcinoma Cells

doi: 10.3390/cancers16132370

Figure Lengend Snippet: SMAD7 knockdown in HCT116 cells reduces the migration rate and the formation of F-ACTIN filaments. ( A ) The HCT116 cells were transfected with either SMAD7 sense or AS for 24 h. Representative images of cell migration captured at time 0 and 24 h by a phase-contrast microscope (10×). The figure is representative of three separate experiments in which similar results were obtained. ( B ) Quantitative analysis showing the percentage of wound margination at 24 h in comparison to that measured at time 0. The values indicate the mean ± SD; the differences were analyzed using a two-tailed Student’s t -test (*** p < 0.001). ( C ) The migration rate was monitored in real-time using the xCELLigence system and data indicate the mean values of three independent experiments. ( D ) The HCT116 cells were transfected as above. Representative confocal microscopy images showing F-ACTIN (red) and DAPI (blue) staining (60×). The figure is representative of three separate experiments in which similar results were obtained. ( E ) Quantitative analysis of the fluorescence intensity in cells cultured as above. The values indicate the mean ± SD; the differences were analyzed using a two-tailed Student’s t -test (*** p < 0.001).

Article Snippet: The 16-well RTCA CIM Plates (Agilent Technologies Inc., Santa Clara, CA, USA) were used to evaluate the cell migration ability in real-time using the xCELLigence System Real-Time Cell Analyzer (ACEA Biosciences, Agilent Technologies Inc., Santa Clara, CA, USA).

Techniques: Knockdown, Migration, Transfection, Microscopy, Comparison, Two Tailed Test, Confocal Microscopy, Staining, Fluorescence, Cell Culture

Journal: Pharmaceuticals

Article Title: Identification of South African Plant-Based Bioactive Compounds as Potential Inhibitors against the SARS-CoV-2 Receptor

doi: 10.3390/ph17070821

Figure Lengend Snippet: Real-time monitoring of HEK293T cells after treatment with A. annua and A. afra leaves’ extracts at different concentrations ranging from 250 to 7.81 µg/mL using the xCELLigence RTCA analyzer.

Article Snippet: In brief, the xCELLigence instrument (Real-Time Cell Analyzer—RTCA; ACEA Biosciences Inc., San Diego, CA, USA), an impedance technology with RTCA software (version 1.2.1), was utilized to monitor the exposure effects of A. annua and A. afra leaves’ methanol extracts on the viability of HEK293T cells, according to the manufacturer’s instructions [ ].

Techniques:

Journal: MedComm

Article Title: CircMALAT1 promotes cancer stem‐like properties and chemoresistance via regulating Musashi‐2/c‐Myc axis in esophageal squamous cell carcinoma

doi: 10.1002/mco2.612

Figure Lengend Snippet: CircMALAT1 promotes cell proliferation, migration, invasion and impedes apoptosis in ESCC cells. The KYSE150 cells were transfected with circMALAT1 plasmid and corresponding controls. (A) Cell proliferation ability was evaluated by colony formation. (B) Growth ability analysis with the xCELLigence Real‐Time Cell Analyzer (RTCA)‐MP system. (C) Transwell assays were used to assess the migration and invasion abilities of ESCC cells. Scale bar, 100 µm. (D) Wound‐healing assays were performed to measure the migration abilities of ESCC cells. Scale bar, 200 µm. (E) Caspase3/7, caspase8, and caspase9 activity was assessed using the fluorogenic substrate after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. (F) Western blot analysis of apoptosis‐related protein levels after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. (G) Flow cytometry analysis of apoptosis (Annexin V/PI) cells after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. The data are presented as the mean ± SD, *** p < 0.001.

Article Snippet: The ESCC cells' capacity for growth was assessed with the xCELLigence Real‐Time Cell Analyzer (RTCA)‐MP system from Acea Biosciences/Roche Applied Science, as detailed in a previous study.

Techniques: Migration, Transfection, Plasmid Preparation, Activity Assay, Western Blot, Flow Cytometry

Journal: MedComm

Article Title: CircMALAT1 promotes cancer stem‐like properties and chemoresistance via regulating Musashi‐2/c‐Myc axis in esophageal squamous cell carcinoma

doi: 10.1002/mco2.612

Figure Lengend Snippet: Silencing of circMALAT1 inhibits the proliferation, migration, invasion and induces apoptosis in ESCC cells. The KYSE450 cells were transfected with circMALAT1‐specific siRNA and corresponding controls. (A) Cell proliferation ability was evaluated by colony formation. (B) Growth ability analysis with the xCELLigence Real‐Time Cell Analyzer (RTCA)‐MP system. (C) Transwell assays were used to measure the migration and invasion abilities of ESCC cells. Scale bar, 100 µm. (D) Wound‐healing assays were performed to assess the migration abilities of ESCC cells. Scale bar, 200 µm. (E) Caspase3/7, caspase8, and caspase9 activity was assessed using the fluorogenic substrate after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. (F) Western blot analysis of apoptosis‐related protein levels after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. (G) Flow cytometry analysis of apoptosis (Annexin V/PI) cells after the indicated cells were treated with cisplatin (20 µg/mL) for 24 h. The data are presented as the mean ± SD, *** p < 0.001.

Article Snippet: The ESCC cells' capacity for growth was assessed with the xCELLigence Real‐Time Cell Analyzer (RTCA)‐MP system from Acea Biosciences/Roche Applied Science, as detailed in a previous study.

Techniques: Migration, Transfection, Activity Assay, Western Blot, Flow Cytometry