Anti cd3ζ 6b10.2

Manufactured by Santa Cruz Biotechnology

Anti-CD3ζ (6B10.2) is a mouse monoclonal antibody that recognizes the CD3ζ chain, a component of the T cell receptor (TCR) complex. The CD3ζ chain is essential for the assembly and expression of the TCR on the cell surface, as well as for signal transduction upon antigen recognition.

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Lab products found in correlation

Anti lamp1 ab24170, by Abcam (1 mentions) Anti foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions) Anti tcrβ h57 597, by Thermo Fisher Scientific (1 mentions) Anti cd3 ucht1, by Thermo Fisher Scientific (1 mentions) Anti rab11 d4f5, by Cell Signaling Technology (1 mentions) Cytochalasin d ab143484, by Abcam (1 mentions) Anti vdr d 6, by Santa Cruz Biotechnology (1 mentions) Anti p53 9282, by Cell Signaling Technology (1 mentions) Ketoconazole k1003, by Merck Group (1 mentions) Bml dm 100 0001, by Enzo Life Sciences (1 mentions) C4859, by Merck Group (1 mentions) Goat anti mouse crosslinker, by Merck Group (1 mentions) Anti β actin, by Abcam (1 mentions) Anti plc γ1 py783, by Cell Signaling Technology (1 mentions) Anti src py416, by Cell Signaling Technology (1 mentions) Anti erk1 2 ptpy185 187, by Thermo Fisher Scientific (1 mentions) Dylight 680, by Thermo Fisher Scientific (1 mentions) Anti il 12 rβ2 305719, by R&D Systems (1 mentions) Anti gapdh, by Meridian Bioscience (1 mentions) Anti sos1, by Santa Cruz Biotechnology (1 mentions)

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Anti-CD3ζ (4 citations)

4 protocols using anti cd3ζ 6b10.2

Comprehensive T Cell Immunophenotyping

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For the flow cytometry analysis, anti-CD4 (GK1.5), anti-CD8α (53–6.7), anti-CD44 (IM7), anti-CD127 (A7R34), anti-CD69 (FN50), anti-Bcl-2 (BCL/10C4) were purchased from Biolegend. Anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD5 (53–7.3), anti-CD45.1 (A20), anti-CD45.2 (104), mouse lgG1 κ isotype control (556027), anti-CD25 (PC61), Rat lgG2a isotype control (eBRG1) and purified anti-human-CD3 (OKT3) were purchased from BD Bioscience. Anti-Foxp3 (FJK-16s), anti-TCRβ (H57–597) and anti-CD3 (UCHT1) were purchased from eBioscience. Fluo-4,AM (F14217) was purchased from Invitrogen. Streptavidin (NEBN7021S) was purchased from Sigma.
For western analysis, anti-α-tubulin (sc-53029) and anti-Arp3 (sc15390) antibodies were purchased from Santa Cruz Biotechnology. Anti-Arpc2 (ab96779) was purchased from Abcam.
For immunofluorescence analysis, anti-Mouse TCR β-chain-488 (H57-597) and anti-Mouse CD3-488 (17A2) were purchased from Biolegend. Anti-CD3ζ (6B10.2) was purchased from Santa Cruz. Anti-EEA1 (C45B10), anti-Rab5 (C8B1) and anti-Rab11 (D4F5) were purchased from Cell Signaling Technology. Anti-LAMP1 (ab24170) was purchased from Abcam.
Cytochalasin D (ab143484) was purchased from Abcam. Staphylococcal enterotoxin E (ET404) was purchased from Toxin Technology. CELLTRACKER(TM) BLUE CMAC (C2110) was purchased from Invitrogen.

Zhang Y., Shen H., Liu H., Feng H., Liu Y., Zhu X, & Liu X. (2017). Arp2/3 complex controls T cell homeostasis by maintaining surface TCR levels via regulating TCR+ endosome trafficking. Scientific Reports, 7, 8952.

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Vitamin D Metabolite Quantification

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25(OH)D₃ (BML-DM-100-0001) and 1,25(OH)₂D₃ (BML-DM200-0050) were from Enzo Life Sciences, Inc., Ann Arbor, MI. Stock solutions of 2.5 mM 25(OH)D₃ and 2.4 mM 1,25(OH)₂D₃ were prepared in anhydrous (≥99.5%) ethanol and stored at −80°C. To determine 1,25(OH)₂D₃ in the supernatants we used the 1,25-Dihydroxy Vitamin D EIA kit (AC-62F1) from IDS, Tyne and Wear, UK according to the manufacturer's instructions. Antibodies used included anti-VDR (D-6) and anti-CD3ζ (6B10.2) from Santa Cruz Biotechnology, Santa Cruz, CA, anti-p53 (9282) from Cell Signaling Technology, Danvers, MA, anti-GAPDH from (ab9485) from Abcam, Cambridge, MA and HRP-rabbit anti-mouse Ig (P0260) from DAKO, Glostrup, Denmark. Cycloheximide ready made solution 100 mg/ml in DMSO (C4859), phorbol 12-myristate 13-acetate (PMA, P8139), ionomycin (I0634), monensin (M5273), leptomycin B (LMB) (L2913) and ketoconazole (K1003) were from Sigma-Aldrich, St. Louis, MO. A fresh solution of ketoconazole 20 mg/ml in anhydrous ethanol was prepared before each experiment. The proteasome inhibitors lactacystin (426100) and MG-132 (474788) were from Merck Millipore, Nottingham, UK.

Kongsbak M., von Essen M.R., Boding L., Levring T.B., Schjerling P., Lauritsen J.P., Woetmann A., Ødum N., Bonefeld C.M, & Geisler C. (2014). Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells. PLoS ONE, 9(5), e96695.

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NK Cell Activation and Signaling Pathway Analysis

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NKL cells (5×10⁶ cells/condition) electroporated with CD3ζ siRNA or control siRNA were stimulated with 5μg/ml of anti-CD16 (3G8) and 2.5μg/ml of goat anti-mouse crosslinker (EMD Millipore) or 5 ×10⁴ K562 cells for one minute. Stimulation was stopped by washing cells twice with cold PBS. In the case of murine NK cells, 3×10⁶ IL-2 stimulated NK cells were stimulated using 3×10⁴ YAC-1 cells. Total protein was extracted using RIPA buffer plus proteinase and phosphatase inhibitors. For Western blot anti-p-Tyr (PY20, Santa Cruz Biotechnology), anti-CD3ζ (6B10.2, Santa Cruz Biotechnology) and anti-β actin (Abcam) were used. Images were acquired with a FUJI LAS-4000 imager (GE Healthcare Life Sciences) and analyzed using FIJI. Lanes from Western blot of lysates from murine NK cells were cut and pasted to place together with their control.

Suárez-Fueyo A., Bradley S.J., Katsuyama T., Solomon S., Katsuyama E., Kyttaris V.C., Moulton V.R, & Tsokos G.C. (2018). Downregulation of CD3ζ in Natural Killer Cells from Systemic Lupus Erythematosus Patients Confers a Pro-Inflammatory Phenotype. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3077-3086.

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Antibody Immunoblotting and Staining Protocol

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Antibodies used for immunoblotting, cell-surface, and intracellular stains were purchased from commercial sources. The anti-LAT Y191 (C305) was from Millipore. The anti-LCK pY505 (4/Lck (pY505), anti-SLP-76 pY128 (J141-668.36.58), and anti-IL-12Rβ1 (114) were from BD Biosciences. The anti-PLC-γ1 pY783 (polyclonal), anti-p38 pT180/Y182 (3D7), anti-p38 (polyclonal), anti-AKT pT308 (244F9), anti-ZAP-70 pY319 (polyclonal), anti-SRC pY416 (polyclonal), anti-STAT4 (2A2), anti-JNK T183/Y185 (polyclonal), anti-MKK3/MKK6 pS189/S207 (D8E9), and anti-STAT4 pY693 (D2E4) antibodies were purchased from Cell Signaling Technologies. The anti-ERK1/2 pTpY185/187 (polyclonal) was from Invitrogen. The anti-SOS1 (polyclonal) and anti-CD3-ζ (6B10.2) from Santa Cruz Biotechnology. The anti-GAPDH was from Meridian Life Science. The DyLight 800 and DyLight 680 labeled secondary antibodies were obtained from Thermo Scientific. The FITC anti-IFN-γ (4S.B3), APC anti-TNF-α (Mab11), anti-CD3 (OKT3), anti-CD28 (CD28.2), anti-CD2 (RPA-2.10), anti-CD49d (9F10), anti-CD11a (HI111) from BioLegend. Anti-IL-12 Rβ2 (305719) was purchased from R&D Systems. The anti-mouse IgG was from Southern Biotech.

Vacaflores A., Freedman S.N., Chapman N.M, & Houtman J.C. (2016). Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production. Molecular immunology, 81, 1-15.

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