TLR2 antibody CD282, a bivalent human recombinant Fab antibovine antibody (AF 647 CD282 (AbD Serotec)), was prepared at 1/10 dilution in staining buffer. CD14 antibody, a mouse anti‐human antibody cross‐reactive with bovine (PerCP‐Cy5.5 CD14 (BioLegend)), was diluted 1/25 in staining buffer. Twenty‐five microlitres from each diluted antibody was added to 50 μL of blocking buffer containing cells, to have a final concentration of 1/40 for TLR2 and 1/100 for CD14 antibody. For detailed antibody description, see Table S5 included in Document S1 of Supporting Information. Then, tubes were incubated at 4°C for 1 h. After washing cell pellets, those were fixed in 100 μL of 1% paraformaldehyde in D‐PBS for 1 h. Then, 550 μL of staining buffer was added and tubes were left overnight at 4°C until flow cytometry processing of samples.
Percp cy5.5 cd14
Manufactured by BioLegend
PerCP‐Cy5.5 CD14 is a fluorochrome-conjugated antibody that targets the CD14 cell surface receptor. It is designed for flow cytometric analysis.
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2 protocols using percp cy5.5 cd14
1
Bovine TLR2 and CD14 antibody staining
2
Phenotypic Analysis of Freshly Isolated PBMCs
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The phenotype of freshly isolated PBMCs were analyzed by flow cytometry. The following fluorochrome-conjugated mAbs and corresponding isotype control antibodies were used: APC-cy7-CD3 (clone: UCHT1; Biolegend), Percp-cy5.5-CD14 (clone: M5E2; Biolegend), Percp-cy5.5-CD19 (clone: HIB19; Biolegend), APC-CD16 (clone: 3G8; Biolegend), PE-cy7-CD56 (clone: HCD56; Biolegend), PE-CD160 (clone: BY55; Biolegend), PE-IgM (clone: MM-30; Biolegend), Brilliant Violet 421-CD69 (clone: FN50; Biolegend), Brilliant Violet 421-IgG1 (clone: MOPC-21; Biolegend), Alexa Fluor® 488-GLUT1 (clone: 202915; R&D Systems), PE-CD36 (clone: CB38; BD PharMingen™), PE-IgM (clone: G155-228; BD PharMingen™), APC-CD71 (clone: CY1G4; Biolegend), APC-IgG2a (clone: MOPC-173; Biolegend), Brilliant Violet 510-CD98 (clone: UM7F8; BD OptiBuil™), and Brilliant Violet 510-IgG1 (clone: X40; BD OptiBuil™). Fixable Viability Stain 620 (BD Horizon™, USA) was used to evaluate cell viability in all experiments. The phenotypic analysis above was conducted using a BD FACS Canto II Flow Cytometer and analyzed by FlowJo 10.4 software (Ashland, OR, USA).
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