Xcelligence sp platform

Manufactured by Agilent Technologies

The XCelligence SP platform is a cell analysis system designed for real-time monitoring of cell growth, migration, and morphology. It provides label-free and non-invasive measurements of cellular dynamics, allowing researchers to gain valuable insights into cellular behavior without disrupting the cells.

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Lab products found in correlation

Rtca software, by Agilent Technologies (3 mentions) Special 96 well e plate, by Agilent Technologies (3 mentions) 96 well e plate, by Agilent Technologies (1 mentions) Magnisort mouse nk cell enrichment kit, by Thermo Fisher Scientific (1 mentions) Cd49b macs sorting, by Miltenyi Biotec (1 mentions) Rhesus il 2, by MyBioSource (1 mentions) Human il 15, by Thermo Fisher Scientific (1 mentions) Human il 2, by Thermo Fisher Scientific (1 mentions) Human fcr block, by Miltenyi Biotec (1 mentions)

4 protocols using xcelligence sp platform

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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NK and Macrophage Cell Killing Assays

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Deuse T., Hu X., Agbor-Enoh S., Jang M.K., Alawi M., Saygi C., Gravina A., Tediashvili G., Nguyen V.Q., Liu Y., Valantine H., Lanier L.L, & Schrepfer S. (2021). The SIRPα–CD47 immune checkpoint in NK cells. The Journal of Experimental Medicine, 218(3), e20200839.

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Modulation of NK and Macrophage Cytotoxicity

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NK cell killing assays and macrophage killing assays were performed on the XCelligence SP platform and MP platform (ACEA Biosciences). Special 96-well E-plates (ACEA Biosciences) were coated with collagen (Sigma-Aldrich) and 4 × 10⁵ WT, B2M⁻^/⁻CIITA⁻^/⁻, or B2M⁻^/⁻CIITA⁻^/⁻ CD47 tg (pooled or single clones) hiECs were plated in 100 µl cell-specific medium. After the cell index value reached 0.7, human NK cells or human macrophages were added at an E:T ratio of 0.5:1, 0.8:1, or 1:1 with or without 1 ng/ml human IL-2 or human IL-15 (both from PeproTech). In some cases, NK cells were pretreated with human FcR block (catalog no. 130–059-901, concentration 1:5; Miltenyi) for 4 h before addition of target cells. As a negative control, cells were treated with 2% Triton X-100 in cell-specific media (data not shown).
Some wells were pretreated with an anti-CD47 blocking antibody (10 µg/ml, clone B6.H12, mouse IgG1,κ; BioXCell) for 2 h and during the assay or with an anti-SIRPα blocking peptide (2 µg/ml, catalog no. MBS822365; MyBioSource) for 2 h and during the assay. In some cases, cells were treated with an activating anti-LILRB1 antibody (clone GHI/75, mouse IgG2b; Abcam) at a concentration of 1 µg/ml. Data were standardized and analyzed with the RTCA software (ACEA Biosciences).

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Real-time Cytotoxicity Assays on iECs

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Real-time killing assays were performed on the XCelligence SP platform and MP platform (ACEA Biosciences). Special 96-well E-plates (ACEA Biosciences) coated with collagen (Sigma-Aldrich) were used. A total of 4 × 10⁴ mouse iECs were plated in 100 μl of medium. After the cell index reached 0.7, we added 4 × 10⁴ C57BL/6 6 NK cells to ADCC assays or 50 μl of C57BL/6 serum to CDC assays. The following antibodies were used and added after mixing with the NK cells in 50 μl of medium or in the serum as indicated: mouse IgG2a anti-H-2^b (BioXCell, clone AF6-88.5.5.3, BE0121) and humanized anti-CD52 IgG1 (alemtuzumab, ichorbio, ICH4002). Different concentrations ranging from 0.0001 μg ml⁻¹ to 1 μg ml⁻¹ were used. As a negative control, cells were treated with 2% Triton X-100 in medium. Data were standardized and analyzed with RTCA Pro 2.3.2 software (ACEA Biosciences).

Gravina A., Tediashvili G., Rajalingam R., Quandt Z., Deisenroth C., Schrepfer S, & Deuse T. (2023). Protection of cell therapeutics from antibody-mediated killing by CD64 overexpression. Nature Biotechnology, 41(5), 717-727.

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