Whole cell lysates of CAR T cells were generated by lysing 5 × 106 washed cells in 150 µl of RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with 1× cOmplete EDTA free protease inhibitor (Roche) and 0.5 mM sodium vanadate (New England BioLabs), and incubating for 30 minutes on ice. Samples were sonicated at 4°C for 5 minutes to shear DNA. Western blots were then performed on supernatants of centrifuged samples, using an anti-CD3ζ antibody (clone E-3, Santa Cruz Biotech) or an anti-pY142 CD3ζ antibody (clone K25–407.69, BD Bioscience) primary and CleanBlot-IP Detection Reagent (Thermo Scientific) secondary. For analysis of T cells following receptor crosslinking, Mock, CD19.28z CAR and GD2.28z CAR T cells were pre-incubated on ice for 10 minutes with OKT3, the 135.20.1 CD19 CAR anti-idiotype antibody, or the 1A7 GD2 CAR anti-idiotype antibody, respectively (10 µg/mL in PBS). Samples were washed and incubated for 5 minutes on ice with a crosslinking goat anti-mouse F(ab’)2 (catalog number 115-006–071, Jackson ImmunoResearch; 25 µg/ml). Samples were then stimulated at 37°C for 10 minutes, centrifuged and immediately lysed as above.
Anti cd3ζ antibody clone e 3
Manufactured by Santa Cruz Biotechnology
The Anti-CD3ζ antibody (clone E-3) is a mouse monoclonal antibody that recognizes the CD3ζ chain of the T-cell receptor complex. This antibody can be used for the detection and analysis of CD3ζ in various applications, such as Western blotting and flow cytometry.
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2 protocols using anti cd3ζ antibody clone e 3
1
Efficient CAR T Cell Lysis and Activation
2
Efficient CAR T Cell Lysis and Activation
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Whole cell lysates of CAR T cells were generated by lysing 5 × 106 washed cells in 150 µl of RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) with 1× cOmplete EDTA free protease inhibitor (Roche) and 0.5 mM sodium vanadate (New England BioLabs), and incubating for 30 minutes on ice. Samples were sonicated at 4°C for 5 minutes to shear DNA. Western blots were then performed on supernatants of centrifuged samples, using an anti-CD3ζ antibody (clone E-3, Santa Cruz Biotech) or an anti-pY142 CD3ζ antibody (clone K25–407.69, BD Bioscience) primary and CleanBlot-IP Detection Reagent (Thermo Scientific) secondary. For analysis of T cells following receptor crosslinking, Mock, CD19.28z CAR and GD2.28z CAR T cells were pre-incubated on ice for 10 minutes with OKT3, the 135.20.1 CD19 CAR anti-idiotype antibody, or the 1A7 GD2 CAR anti-idiotype antibody, respectively (10 µg/mL in PBS). Samples were washed and incubated for 5 minutes on ice with a crosslinking goat anti-mouse F(ab’)2 (catalog number 115-006–071, Jackson ImmunoResearch; 25 µg/ml). Samples were then stimulated at 37°C for 10 minutes, centrifuged and immediately lysed as above.
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