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CD6-RFP Jurkat cell line

Invented by Alessandra Cambi
Invented at Radboud UMC
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CD6 and TCR/CD3 in Jurkat T cells co-localize upon ligation through micropatterned antibody spots. (A,B) Wildtype Jurkat T cells were seeded on micropatterned substrates containing 100, 10, or 1% αCD3 spots, 100% αCD6 spots or 100% fibronectin (FN) spots, surrounded by fibronectin, fixed after 15 min and stained for phospho-tyrosine. All spots were labeled with Alexa647, phospho-tyrosine was labeled with Alexa488. Representative confocal images are shown in (A), quantification of phospho-tyrosine intensity in the spot area of n = 10 cells per condition is shown in (B). Bars represent mean with SD. Statistical significance was tested with one-way ANOVA with post-hoc Tukey's Multiple Comparison test. (C,D) CD6-RFP Jurkat T cells were seeded on micropatterned substrates containing 100, 10, or 1% αCD3 spots, or 100% αCD6 spots, surrounded by fibronectin, and fixed after 15 min. All spots were labeled with Alexa647. Representative confocal images are shown in (C), quantification of CD6 enrichment of n = 10 cells per condition is shown in (D). CD6 enrichment is defined as the ratio between CD6-RFP intensity of parts of the cell on the spot vs. part of the cell covering the surrounding fibronectin. Bars represent mean with SD. Statistical significance was tested with Kruskal–Wallis test with post-hoc Dunn's Multiple Comparison test. (E,F) Wildtype Jurkat cells were seeded on micropatterned substrates containing 100, 10, or 1% αCD3 spots, or 100% αCD6 spots, surrounded by fibronectin, fixed after 15 min and stained for CD3. All spots were labeled with Alexa647, CD3 was labeled with TexasRed. Representative confocal images are shown in (E), quantification of CD3 enrichment of n = 10 cells per condition is shown in (F). Bars represent mean with SD. CD3 enrichment is defined as the ratio between CD3 intensity of parts of the cell on the spot vs. part of the cell covering the surrounding fibronectin. Statistical significance was tested with Kruskal–Wallis test with post-hoc Dunn's Multiple Comparison test. Scale bars represent 10 μm; *p < 0.05; **p < 0.01; ***p < 0.001. Taken from Meddens MBM, Mennens SFB, Celikkol FB, Te Riet J, Kanger JS, Joosten B, Witsenburg JJ, Brock R, Figdor CG, Cambi A. Biophysical Characterization of CD6-TCR/CD3 Interplay in T Cells. Front Immunol. 2018 Oct 9;9:2333. doi: 10.3389/fimmu.2018.02333. PMID: 30356797; PMCID: PMC6189472.
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Catalogue Number 160713
Antigen/Gene or Protein Targets Cluster of Differentiation 6 labelled with RFP, CD6-RFP
Parental Line Jurkat E6.1
Synonyms CD6
Host Human
Tissue Blood
Disease Keywords Acute T cell Leukemia
Relevance Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts.
Production Details Jurkat cell lines stably expressing CD6-RFP were obtained by electroporation using the Neon Transfection System for Electroporation (Invitrogen) according to the manufacturer's guidelines. Shortly, 5*105 Jurkat cells were transfected at 1325 Volt (10 ms, 3 pulses) with 3 μg of DNA in 100 μl Resuspension buffer. After transfection cells were seeded in 2 ml of 1640 RPMI with 10% FCS and 1% U-glut. Antibiotics were added after 3 h. Stable cell lines were sorted on RFP expression on a FACSAria cell sorter (BD Biosciences), and cells were maintained in complete RPMI medium as described above, additionally supplemented with 500 ng/ml geneticin (G418, Gibco).
Conditional No
Research Area Adhesion, Cancer, Cell Signaling & Signal Transduction, Immunology
Recommended Growing Conditions 1640 RPMI (PAA) supplemented with 10% Fetal Calf Serum, 1 mM Ultra-glutamine and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B). 500 ng/ml geneticin.

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Meddens et al. 2018. Front Immunol. 9:2333. PMID: 30356797.

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References: 1 entry

Meddens et al. 2018. Front Immunol. 9:2333. PMID: 30356797.

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Inventor Information

Inventors

Alessandra Cambi

Alessandra Cambi

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Invented at

Radboud UMC
Radboud UMC

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