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Phospho lck

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Phospho-LCK is a laboratory reagent used in the detection and quantification of the phosphorylated form of the LCK (Lymphocyte-Specific Protein Tyrosine Kinase) protein. LCK is an important signaling molecule in T cell activation and development. The Phospho-LCK reagent can be used in various analytical techniques, such as Western blotting and ELISA, to measure the levels of phosphorylated LCK in biological samples.

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2 protocols using phospho lck

1

Phosphoflow Analysis of PBMC Signaling

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PBMCs from healthy patients were treated with each drug for 2 hours and stimulated with 3.3 mM H2O2 for 10 minutes at 37°C. The cells were then fixed with 1.6% paraformaldehyde solution for 10 minutes at 37°C and permeabilized with 100% methanol overnight at –80°C. Cells were stained with antibodies against phospho-LCK (#558552; BD Biosciences, Franklin Lakes, NJ), phospho-SRC (#560096; BD Biosciences), cleaved Poly(ADP-ribose) polymerase (PARP) (#560640; BD Biosciences) and CD3 (#562426; BD Biosciences). A similar assay was previously described for T-cell phosphoflow analysis using CD3/CD28 and H2O2 stimuli (38 (link),39 (link)). Data were acquired on a FACSVerse flow cytometer and analyzed with FCS Express (version 4; BD Biosciences).
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2

Multiparametric Flow Cytometry Assay

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For flow cytometry, fluorochrome-conjugated antibodies against the following antigens were used: CD56 (clone NCAM16.2; conjugated to BV605) (B159; APC), CD3 (SK7; BUV395) (HIT3a; PE), CD107a (H4A3; APC), IFN-γ (B27; PerCP/Cy5.5), TNF (MAb11; PE), CD69 (FN50; BV421 and BV711), CD11a (HI111; BV711), NKG2D (149810; PE-CF594), NKp30 (p30-15; BV421), TACTILE (6F9; BV711), TRAIL (RIK-2; BV421), BTLA (J168-540; PE-CF594), Phospho-Lck (4/LCK-Y505; AF647), Phospho-LAT (J96-1238.58.93; PE), Phospho-SLP-76 (J141-668.36.58; PE), Phospho-MEK1/2 (O24-836; PE), Phospho-ERK1/2 (20A; BV421), Phospho-SHP-2 (L99-921; AF647) (all BD Biosciences), CD160 (BY55; PE/Cy7), NKp46 (9E2; PE/Cy7),NKp44 (P44-8; PE), DNAM-1 (11A8; APC), 2B4 (C1.7; PerCP/Cy5.5), NKp80 (5D12; PE) (all BioLegend), NKG2C (134591; APC) (R&D Systems), and VZV gE:gI (SG1-1; conjugated in-house to DyLight 488) (Meridian Life Science). Matched isotype control antibodies were also used where appropriate.
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