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CD6-LifeAct-GFP Jurkat cell line

CD6-LifeAct-GFP Jurkat cell line

Invented by Alessandra Cambi

Invented at Radboud UMC

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$CD6 is co-transported with TCR/CD3 in microclusters toward the center of the immunological synapse formed on αCD3-containing SLBs. (A,B) Wildtype Jurkat T cells were seeded for 30 min on SLBs containing ATTO647-conjugated αCD3, and subsequently fixed and stained for immunological synapse markers. Representative TIRF microscopy images for phospho-tyrosine staining and LFA-1 staining (both labeled with Alexa488) are shown in (A,B) respectively. (C) LifeAct-GFP Jurkat T cells were seeded for 30 min on SLBs containing ATTO647-conjugated αCD3 and imaged using live cell imaging TIRF microscopy. Representative images are shown in (C). (D–G) CD6-GFP Jurkat T cells were imaged while settling on a SLB containing ATTO647-conjugated αCD3, using TIRF microscopy live cell imaging. Snap shots of every minute of the time lapse are shown in (D). Snap shots at time point 3 min, including a zoom-in of CD6-GFP and αCD3 microclusters are shown in (E). Kymograph of the line (indicated in the Merge image at time point 4 min in D) during 3:45 to 6:12 min of the imaging period is shown in (F). Co-localization over time starting at time point 2 min, represented as Mander's coefficient M1 (fraction of CD6-GFP overlapping with αCD3) in the central zoomed-in region indicated in (E) is shown in (G). Scale bars represent 10 μm. Taken from Meddens MBM, Mennens SFB, Celikkol FB, Te Riet J, Kanger JS, Joosten B, Witsenburg JJ, Brock R, Figdor CG, Cambi A. Biophysical Characterization of CD6-TCR/CD3 Interplay in T Cells. Front Immunol. 2018 Oct 9;9:2333. doi: 10.3389/fimmu.2018.02333. PMID: 30356797; PMCID: PMC6189472.$

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Catalogue Number	160715
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-LifeAct-YFP 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 YFP 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 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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