For western blotting, antibodies were used as follows: TRF2 (1:500, Cell Signaling Technology, 13136), p16 (1:1000, Proteintech, 10883-1-AP), p21 Waf1/Cip1 (1:1000, Cell Signaling Technology, 2947), γ-H2AX (ser139) (1:500, Cell Signaling Technology, 9718), 53BP1 (1:1000, Novus, NB100-304), p53 (1:1000, Cell Signaling Technologies, 2524), P-p53 (1:1000, Cell Signaling Technologies, 9284), Myc (1:1000, Cell signaling, Technology 2276), P-TBK1 (Ser 172) (1:1000, Cell Signaling Technology, 5483), TBK1 (1:1000, Cell Signaling Technology, 3013), NF-κB P-p65 (Ser 536) (1:1000, Cell Signaling Technology, 3033), NF-κB p65 (1:1000, Cell Signaling Technology, 8242), NF-κB1 p105/p50 (1:1000, Cell Signaling Technology, 3035), cGAS (1:1000, Cell Signaling Technology, 15102), Actin (1:10 000, Sigma-Aldrich, A5-441), anti-rabbit HRP (1:3000, Cell Signaling Technology, 7074), anti-mouse HRP (1:3000, Cell Signaling Technology, 7076). For immunostaining: γ-H2AX (JBW301) (1:500, Merck, 05-636), H3K9me3 (1:400, Abcam, ab8898), 53BP1 (1:500, Novus, NB100-304), αSMA (Biotin) (1:500, Abcam, ab125057), CD45 (Alexa Fluor 647) (1:50, Biolegend, 103123), CD3 (Alexa Fluor 647) (1:50, Biolegend, 100209), CD68 (Alexa Fluor 647) (1:50, Biolegend, 137001), ICAM-1 (1:50, Biolegend, 116102), VCAM-1 (1:50, Biolegend, 105702).
Cd3 alexa fluor 647
Manufactured by BioLegend
CD3 (Alexa Fluor 647) is a fluorescently-labeled antibody that binds to the CD3 surface antigen. Alexa Fluor 647 is the fluorescent dye conjugated to the CD3 antibody. This product is intended for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe cf594, by BD (2 mentions)
Cd19 bv711, by BD (2 mentions)
Cd8a apc efluor 780, by Thermo Fisher Scientific (2 mentions)
P21 waf1 cip1, by Cell Signaling Technology (1 mentions)
γh2ax jbw301, by Merck Group (1 mentions)
P tbk1 ser172, by Cell Signaling Technology (1 mentions)
Icam 1, by BioLegend (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Nf κb1 p105 p50, by Cell Signaling Technology (1 mentions)
Cd45 alexa fluor 647, by BioLegend (1 mentions)
H3k9me3, by Abcam (1 mentions)
γh2ax ser139, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa sorp, by BD (1 mentions)
Cd14 bv605, by BioLegend (1 mentions)
Cd16pe, by R&D Systems (1 mentions)
3 protocols using cd3 alexa fluor 647
1
Western Blotting and Immunostaining Assays
2
Isolation and Flow Cytometry of PBMCs
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The isolation of peripheral blood mononuclear cells (PBMCs) and flow-based transmigration assays were performed in a 3D BioFlux flow chamber device (Fluxion Bioscience, San Diego, CA) as previously described.30 (link) The migrated cells were enumerated by a hemocytometer and then normalized to migrated cell numbers determined by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature and washed in phosphate buffered saline + 0.1 mM ethylenediaminetetraacetic acid, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 eFluor450, CD8a APC-eFluor780 (eBioscience, San Diego, CA), CD3Alexa Fluor 647 (BioLegend, San Diego, CA), CD19 BV711, and CD4 PE-CF594 (BD Biosciences). Data were acquired using BD FACSCanto II (BD Biosciences) and analyzed by FlowJo software (v.10.4.1; Treestar, Ashland, OR).
3
Multiparameter Flow Cytometry Immunophenotyping
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Input and migrated cells were analyzed by flow cytometry. After collection, cells were fixed for 10 minutes in 1% paraformaldehyde at room temperature, washed in PBS/0.1 mM EDTA, followed by blocking in mouse IgG. Cells were labeled with anti-human CD45 efluor450, CD8a APC-efluor780 (eBiosciences, San Diego, CA), CD3 Alexa Fluor 647, CD14 BV605 (BioLegend, San Diego, CA), CD19 BV711, CD4 PE-CF594 (BD Biosciences), and CD16 PE (R&D Systems). Data were acquired on a BD LSRFortessa SORP flow cytometer running Diva6, and analyzed in FlowJo 9.7.5 (Tree Star, Ashland, OR).
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