After treatment, BMDMs were fixed with 4% (w/v) paraformaldehyde (PFA), blocked with 5% (w/v) bovine serum albumin (BSA), and then incubated with FITC-conjugated F4/80 antibody (11-4801-82, eBioscience, USA), APC-conjugated CD206 antibody (17-2061-82, eBioscience, USA), and PE-conjugated CD86 antibody (12-0862-82, eBioscience, USA) for 30 min. The candidate cells were detected using a BD FACS Caliber flow cytometer and analyzed using FlowJo v10.0 software. F4/80+ cells were identified as macrophages (Figure S1 ), and the expression levels of CD86 and CD206 were detected to evaluate the M1 and M2 polarization states of BMDMs.
Apc conjugated cd206 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APC-conjugated CD206 antibody is a laboratory reagent used for the detection and analysis of the CD206 protein, also known as the macrophage mannose receptor, in cell samples. It is conjugated with the fluorescent dye allophycocyanin (APC) for visualization and quantification purposes during flow cytometry or other immunoassay experiments.
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Lab products found in correlation
Pe conjugated cd86 antibody, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated f4 80 antibody, by Thermo Fisher Scientific (2 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (2 mentions)
Facsverse, by BD (2 mentions)
Pe cyanine7 conjugated inos antibody, by Thermo Fisher Scientific (2 mentions)
Facs caliber, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
4 protocols using apc conjugated cd206 antibody
1
Macrophage Phenotyping by Flow Cytometry
2
Macrophage Polarization Evaluation
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After treatment, BMDMs were xed with 4% paraformaldehyde (PFA), blocked with 5% bovine serum albumin (BSA), and then incubated with FITC-conjugated F4/80 antibody (eBioscience, USA), APCconjugated CD206 antibody (eBioscience), and PE-conjugated CD86 antibody (eBioscience) for 30 min. The candidate cells were detected using a BD FACS Caliber ow cytometer and analyzed using FlowJo v10.0 software. F4/80 + cells were identi ed as macrophages, and the expression levels of CD86 and CD206 were detected to evaluate the M1 and M2 polarization states of BMDMs.
3
Characterizing Macrophage Polarization
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BMDMs were stimulated by wear particles for 24 h and then treated with TrypLE Express Enzyme (12604021, Thermo Fisher Scientific) to detach cells from the plates. They were then washed with Perm/Wash buffer, suspended in PBS and analyzed using flow cytometry (BD FACSVerse, BD Biosciences). After centrifugation at 1,000 rpm for 5 min, BMDMs were sequentially incubated with APC-conjugated CD206 antibody (0.25ug/test, 17-2061-82, Thermo Fisher Scientific) and PE-cyanine7-conjugated iNOS antibody (0.06ug/test, 25-5920-82, Thermo Fisher Scientific). The isotype controls used were APC-conjugated rat IgG2b, κ (17-4031-82, Thermo Fisher Scientific) and PE-cyanine7-conjugated rat IgG2a, κ (25-4321-82, Thermo Fisher Scientific).
4
Phenotypic Analysis of BMDMs
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The BMDMs were seeded in 6-well plates at a concentration of 5 × 10 5 cells per well. Twenty-four hours after seeding, the BMDMs were stimulated by TiPs for 24 h and then treated with TrypLE Express Enzyme (12604021, Thermo Fisher Scientific) to remove them from the plates. Then, the BMDMs were centrifuged at 1000 rpm for 5 min. Following our recent study protocol, 12 after washing in PBS, the BMDMs were incubated with APC-conjugated CD206 antibody (17-2061-82, Thermo Fisher Scientific) at 20 °C for 20 min in the dark. Then, the BMDMs were washed with PBS and incubated with a fixation buffer (00-8222-49, Thermo Fisher Scientific) at 20 °C for 20 min in the dark. After washing with Perm/Wash buffer (554723, BD Biosciences), the BMDMs were incubated with PE-cyanine7-conjugated iNOS antibody (25-5920-82, Thermo Fisher Scientific) at 20 °C for 20 min in the dark. The isotype controls used were APC-conjugated rat IgG2b,κ (17-4031-82, Thermo Fisher Scientific) and PE-cyanine7-conjugated rat IgG2a,κ (25-4321-82, Thermo Fisher Scientific). After washing with the Perm/Wash buffer, the BMDMs were suspended in PBS and analyzed using flow cytometry (BD FACSVerse, BD Biosciences).
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