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CD6-GFP Jurkat cell line

Invented by Alessandra Cambi
Invented at Radboud UMC
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Disruption of actin polymerization inhibits TCR/CD3 and CD6 co-transport toward the cSMAC of the immunological synapse on αCD3-containing SLBs. (A,B) CD6-GFP Jurkat T cells, either untreated or pretreated with 0.5 μM Cytochalasin D for 15 min, were seeded on a SLB containing ATTO647-conjugated αCD3. Twenty minutes after seeding CD6-GFP and αCD3 dynamics were imaged using TIRF microscopy live cell imaging. Snap shots of every 30 s of the time lapse are shown in (A). Kymograph of the line (indicated in the Merge image at time point 0 min in A) during the whole imaging period (4.5 min) is shown in (B). (C) Wildtype Jurkat T cells, either untreated or pretreated with 0.5 μM Cytochalasin D for 15 min, were seeded for 30 min on SLBs containing ATTO647-conjugated αCD3, and subsequently fixed. Widefield microscopy was performed and cells (>44 cells per condition; three independent experiments) were scored for synapse formation based on identification of cells by brightfield displaying αCD3 positive cluster formation in a lipid bilayer (DiI) positive area. Average percentages of cells forming an immunological synapse are represented in (C). Bars represent mean with SD. Statistical significance was tested with paired t-test. Scale bar represents 10 μm; *p < 0.05. 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 160714
Antigen/Gene or Protein Targets Cluster of Differentiation 6 labelled with GFP, CD6-GFP
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-GFP 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 GFP 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, Cell Signaling & Signal Transduction, Immunology
Recommended Growing Conditions 1640 RPMI 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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