96 well e plate

Manufactured by Agilent Technologies

Sourced in United States

The Agilent 96-well E-plates are specialized lab equipment used for cell-based assays. They provide a platform for monitoring cell proliferation, migration, and viability in real-time. The E-plates incorporate gold electrodes at the bottom of each well, enabling electrical impedance measurements to track changes in cell status.

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38 protocols using 96 well e plate

Measuring CAR T Cell Cytotoxicity and Repeat Killing

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The xCELLigence real-time cell analyzer (RTCA) MP instrument (Agilent Technologies, Santa Clara, CA, USA) was used to assess CAR T cell cytotoxicity and repeat killing capacity. All assays were performed in triplicate and without the addition of exogenous cytokines. First, 30,000 LM7 cells in complete RPMI were added to each well of a 96-well E-Plate (Agilent). After LM7 cells adhered to the E-Plate for approximately 24 h and reached a cell index (relative cell impedance) plateau, 15,000 T cells in complete RPMI were added. LM7 cells alone served as negative controls. The cell index was monitored every 15 min for 3 days and normalized to the maximum cell index value immediately prior to T cell plating. Percent cytotoxicity was calculated using the RTCA Software Pro immunotherapy module (Agilent).⁵² (link) For repeat killing assays, 72 h after T cell plating, media and T cells were gently removed to avoid disrupting adherent LM7 cells and plated on 30,000 fresh LM7 cells adhered to a new 96-well E-Plate. Repeat cytolysis was assessed until T cells stopped killing, defined by no CAR T cell killing greater than 50% of LM7 target cells, or over a maximum of five total stimulations per donor.

Nguyen P., Okeke E., Clay M., Haydar D., Justice J., O’Reilly C., Pruett-Miller S., Papizan J., Moore J., Zhou S., Throm R., Krenciute G., Gottschalk S, & DeRenzo C. (2020). Route of 41BB/41BBL Costimulation Determines Effector Function of B7-H3-CAR.CD28ζ T Cells. Molecular Therapy Oncolytics, 18, 202-214.

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NK Cell Cytotoxicity Assay with Engineered Endothelial Cells

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Mouse NK cells were isolated from fresh BALB/c or C57BL/6 spleens 18 h after poly I:C injection (100 μg intraperitoneally). After red cell lysis, NK cells were purified with MagniSort Mouse NK cell Enrichment Kit (Invitrogen), followed by CD49b MACS-sorting (Miltenyi). This cell population was highly selected for NK cells with a purity of >9%. Human NK cells from PBMCs were purchased from StemCell Technologies containing >99% NK cells.
NK cell killing assays were performed on the XCelligence SP platform (ACEA BioSciences). 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma-Aldrich) and 4 × 10⁵ WT, B2m^−/−Ciita^−/−, or B2m^−/−Ciita^−/− Cd47 tg miECs or WT, B2M^−/−CIITA^−/− or B2M^−/−CIITA^−/− CD47 tg hiECs were plated in 100 μl cell-specific media containing 1 ng ml⁻¹ mouse or human IL-2 (Peprotech). After the Cell Index value reached 0.7, NK cells were added with an effector cell / target cell (E/T) ratio of 0.5/1, 0.8/1 or 1/1. As a negative control, cell treated with 2% Triton X100 was used. Some wells were pretreated with mouse Cd47 or human CD47-blocking antibody (BioXCell) with 10 μg ml⁻¹ media for 2 h. Data were standardized and analyzed with the RTCA software (ACEA).

Deuse T., Hu X., Gravina A., Wang D., Tediashvili G., De C., Thayer W.O., Wahl A., Garcia J.V., Reichenspurner H., Davis M.M., Lanier L.L, & Schrepfer S. (2019). Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nature biotechnology, 37(3), 252-258.

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Real-Time Cell Cytolysis Assay

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Adherent target cells (HT29, HCT116, and LoVo) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 10⁴ cells per well and monitored in culture overnight using impedance-based real-time cell analysis (RTCA) xCELLigence system (Acea Bio). The next day, the media was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 10⁵ effector cells (CAR-T cells or non-transduced T cells), in triplicate. The cells in the E-plates were monitored for another 24–48 h with the RTCA system, and impedance was plotted over time. Cytolysis was calculated as follows: (impedance of target cells without effector cells-impedance of target cells with effector cells) × 100/impedance of target cells without effector cells.

Sureban S.M., Berahovich R., Zhou H., Xu S., Wu L., Ding K., May R., Qu D., Bannerman-Menson E., Golubovskaya V, & Houchen C.W. (2019). DCLK1 Monoclonal Antibody-Based CAR-T Cells as a Novel Treatment Strategy against Human Colorectal Cancers. Cancers, 12(1), 54.

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Real-Time Cytotoxicity Assay for CAR-T Cells

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Adherent target cells (CHO or CHO-BCMA) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 10⁴ cells per well and monitored in culture overnight with the impedance-based real-time cell analysis (RTCA) xCELLigence system (Acea Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 10⁵ effector cells (CAR-T cells or non-transduced T cells), in triplicate. The cells in the E-plates were monitored for another one to two days with the RTCA system, and impedance was plotted over time. Cytolysis was calculated as (impedance of target cells without effector cells−impedance of target cells with effector cells) × 100/impedance of target cells without effector cells.

Berahovich R., Zhou H., Xu S., Wei Y., Guan J., Guan J., Harto H., Fu S., Yang K., Zhu S., Li L., Wu L, & Golubovskaya V. (2018). CAR-T Cells Based on Novel BCMA Monoclonal Antibody Block Multiple Myeloma Cell Growth. Cancers, 10(9), 323.

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Quantifying Cytotoxicity of CAR-T Cells

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One day prior to co-culture with T cells or PBMC, Huh7 or Huh7S cells (4x10⁴ cells/well) were seeded in 96-well E-plates (ACEA Biosciences) to quantify cell viability, or in a conventional 96-well cell culture plate (TPP) to measure cytokine secretion, using 200 µl of culture medium/well. After removing 100 μl of medium, either PBMC combined with the indicated antibodies or S-CAR transduced T cells were added onto target cells in 100 μl at the indicated effector to target (E:T) ratio. Cell culture medium was supplemented with 10% FCS, L-glutamine (2 mM), 1% non-essential amino acids and sodium pyruvate, penicillin (100 U/mL), streptomycin (100 mg/mL), HEPES (10 mM), and gentamycin (16,6 mg/ml) (all from ThermoFisher Scientific). medium at indicated effector to target (E:T) ratios. Molarity given refers to the total antibody concentration used. The co-culture was maintained for 120 hours and cell viability was measured employing an xCELLigence RTCA SP device (ACEA Biosciences).

Debelec-Butuner B., Quitt O., Schreiber S., Momburg F., Wisskirchen K, & Protzer U. (2022). Activation of distinct antiviral T-cell immunity: A comparison of bi- and trispecific T-cell engager antibodies with a chimeric antigen receptor targeting HBV envelope proteins. Frontiers in Immunology, 13, 1029214.

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Real-Time Cell Cytolysis Assay

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Example 6

Adherent target cells were seeded into 96-well E-plates (Acea Biosciences, San Diego, Calif.) at 1×10⁴cells per well and monitored in culture overnight with the impedance-based real-time cell analysis (RTCA) iCELLigence system (Acea Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS±1×10⁵effector cells (CAR-T cells or non-transduced T cells), in triplicate. The cells in the E-plates were monitored for another 2 days with the RTCA system, and impedance was plotted over time. Cytolysis was calculated as (impedance of target cells without effector cells—impedance of target cells with effector cells)×100/impedance of target cells without effector cells.

US11332513B2. Chimeric antigen receptors having GITR intracellular domain as co-stimulatory domain (2022-05-17). ProMab Biotechnologies, Inc. [US], Forevertek Biotechnology Co., Ltd [CN], The General Hospital Corporation [US]. Inventors: Lijun Wu [US], Marcela V. Maus [US], Vita Golubovskaya [US].

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Real-Time Cytotoxicity Evaluation of CAR-T Cells

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Adherent target cells (CHO-CD37; CHO; Hela-CD37 or Hela) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA, USA) at 1 × 10⁴ cells per well and monitored in culture overnight with the impedance-based real-time cell analysis (RTCA) xCELLigence system (Acea Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 × 10⁵ effector cells (CAR-T cells or non-transduced T cells) in triplicate. The cells in the E-plates were monitored for another 24–48 h with the RTCA system, and impedance was plotted over time. Cytotoxicity was calculated as (impedance of target cells without effector cells—impedance of target cells with effector cells) × 100/impedance of target cells without effector cells.

Golubovskaya V., Zhou H., Li F., Valentine M., Sun J., Berahovich R., Xu S., Quintanilla M., Ma M.C., Sienkiewicz J., Huang Y, & Wu L. (2021). Novel CD37, Humanized CD37 and Bi-Specific Humanized CD37-CD19 CAR-T Cells Specifically Target Lymphoma. Cancers, 13(5), 981.

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Kinetic Growth Analysis of Cell Lines

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Cells were plated into 96 well E-plates (ACEA Biosciences, San Diego, CA, USA) and grown in a xCELLigence-RCTA SP system (ACEA Biosciences, San Diego, CA, USA). Cells were seeded in quadruplicates for all conditions. In addition, wells with only media were used as a control. The measurement was taken every 10 min for 120 h. We fitted the growth curve using a logistic function with three characteristic parameters: the length of lag phase, the generation time and the maximum cell index.

Bardales J.A., Wieser E., Kawaji H., Murakawa Y, & Darzacq X. (2018). Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers. Genes, 9(6), 270.

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Cytotoxicity Assay for CAR-T Cell Evaluation

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Adherent target cells were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA) at 1.5 × 10⁴ cells per well (HeLa-CD19), 3 × 10⁴ cells per well (HeLa), or 4 × 10⁴ cells per well (CHO-CD22, CHO-BCMA), and monitored in the normal incubator or hypoxia chamber overnight with the xCELLigence impedance-based RTCA system (Acea Biosciences). The next day, the medium was removed and replaced with normal or equilibrated RPMI-1640 medium containing 10% FBS ± CAR-T cells or non-transduced T cells at an E:T ratio of 10:1, in triplicate. The cells in the E-plates were monitored for another 20–24 h with the RTCA system, and impedance (normalized to the time of effector cell addition) was plotted over time. Cytotoxicity was calculated as the percentage (X − Y) × 100/X, where X = normalized impedance of target cells without effector cells and Y = normalized impedance of target cells with effector cells. For hypoxic RTCA assays, target cells were cultured in the hypoxia chamber for three days before use.

Berahovich R., Liu X., Zhou H., Tsadik E., Xu S., Golubovskaya V, & Wu L. (2019). Hypoxia Selectively Impairs CAR-T Cells In Vitro. Cancers, 11(5), 602.

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Cell Proliferation Assay Protocols

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HPDE cell proliferation was measured 72h post-transfection using the Cell Counting Kit-8 (CCK-8) kit (Dojindo) as described [24 (link), 41 (link)]. MiaPaCa-2 proliferation was measured on an xCELLigence platform (ACEA Biosciences). Cells were seeded 72h post-transfection at 3000 cells/well into 96-well E-Plates (ACEA Biosciences), then transferred into an xCELLigence in a humidified 37^oC chamber in 5% CO₂. Cell Index (Proliferation) was measured hourly for 48h.

McCarroll J.A., Sharbeen G., Liu J., Youkhana J., Goldstein D., McCarthy N., Limbri L.F., Dischl D., Ceyhan G.O., Erkan M., Johns A.L., Biankin A.V., Kavallaris M, & Phillips P.A. (2014). βIII-Tubulin: A novel mediator of chemoresistance and metastases in pancreatic cancer. Oncotarget, 6(4), 2235-2249.

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