BMDMs were plated in a 12-well plate (Corning), 0.25 × 106 cell/well, and polarized to M1 macrophages in IMDM. Nontreated BMDMs were cultured as M0 macrophages. Cells were washed with PBS and collected via centrifugation at 250× g, 4 °C for 5 min. Cells were washed with a washing buffer (PBS with 2% FCS) and centrifuged at 350× g, 4 °C for 5 min. The supernatant was discarded, and the Fc receptors were blocked by 10 min incubation on ice in a blocking buffer (PBS with 2% FCS, 20% 2.4G2 supernatant, 3% NRS, and 5% normal Syrian hamster serum). Cells were incubated with fluorochrome-labeled monoclonal antibodies in a blocking buffer for 20 min on ice. Anti-CD11b PE-Cyanine7 (eBioscience, San Diego, CA, USA) and anti-CD86 APC-R700 (BD Biosciences) antibodies were used for flow cytometry analysis. Cells were washed with a washing buffer and fixed in 2% paraformaldehyde (PFA) at RT for 10 min. Samples were measured with a BD FACSLyric flow cytometer (BD Biosciences), and data were subsequently analyzed with FlowJo 7.6.5. software (Tree Star, Ashland, OR, USA).
Anti cd86 apc r700
Manufactured by BD
Sourced in United States
Anti-CD86-APC-R700 is a fluorochrome-conjugated monoclonal antibody used for the detection and analysis of CD86-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facslyric flow cytometer, by BD (2 mentions)
12 well plate, by Corning (1 mentions)
Anti cd11b pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti cd11b apc, by BioLegend (1 mentions)
Anti f4 80 apc antibody, by BioLegend (1 mentions)
Bv421 anti siglec f, by BD (1 mentions)
Anti cd63 pe, by BD (1 mentions)
Trustain fcx plus anti mouse cd16 32, by BioLegend (1 mentions)
Anti cd80 fitc, by BD (1 mentions)
Anti cd40 pe, by BD (1 mentions)
Anti cd45 pe, by BD (1 mentions)
Adult brain dissociation kit, by Miltenyi Biotec (1 mentions)
Aurora flow cytometer, by Cytek (1 mentions)
Zombie nir fixable viability dye, by BioLegend (1 mentions)
4 protocols using anti cd86 apc r700
1
Polarization of Murine Bone Marrow-Derived Macrophages
2
Multiparameter Flow Cytometry of Adipose SVF
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Stromal vascular fraction (SVF) cells were harvested from the inguinal AT, and to block Fc receptors, they were incubated with FC block (Cat # 156604, TruStain FcX PLUS, anti-mouse CD16/32, BioLegend, London, UK) for 10 minutes. For the macrophages panel, cells were stained with anti-CD86-APC-R700 (Cat # 565479, BD Biosciences, San Jose, USA), anti-F4/80-APC antibody (Cat # 123116, BioLegend), and anti-CD11b PE-Cy7 (Cat # 25-0112-81, eBioscience, San Diego, USA). For the eosinophils panels, anti-Siglec-F BV421 (Cat # 562681, BD Biosciences), anti-CD11b APC (Cat # 101212, BioLegend), anti-CD63 PE(Cat# 564222, BD Biosciences), and anti-CD193 (CCR3) Alexa 647 (Cat# 144508, BioLegend) were used. After staining, cells were washed with a washing buffer (2% FCS in PBS) and analyzed using a BD FACSLyric™ flow cytometer (BD Biosciences).
3
Evaluation of Immune Cell Activation
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The An@AuNPs at a dose of 10 μg/mL was incubated with 5 × 105 macrophages/well or BMDCs/well for 12 h. The macrophages or BMDCs stained with anti-CD86-APC-R700 (BD, cat. no. 565479), anti-CD80-FITC (BD, cat. No. 561954), and anti-CD40-PE (BD, cat. no. 553791) antibody were analyzed via flow cytometric examination.
4
Intravascular Myeloid Cell Identification in Mouse Brain
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Mice were intravenously injected while alive with anti-CD45-PE (BD Biosciences) (3 µg in 100 µl of sterile PBS) to discriminate between intravascular myeloid cells and intraparenchymal myeloid cells [99 (link)]. After sacrifice, brains were collected and processed into a single-cell suspension using adult brain dissociation kit (Miltenyi Biotec, catalog no. 130–107-677) as previously described [31 (link)]. Cells were then washed with cold PBS before staining with live/dead Zombie NIR fixable viability dye (BioLegend) for 30 min at 4 °C. Cells were then washed twice with PBS supplemented with 1% heat-inactivated FBS (1% FBS) (FACS buffer) and resuspended in Fc Block (clone 93) (eBioscience) at 4 °C for 5 min before surface staining with a mixture of the following Abs for 20 min at 4 °C: anti-CD86-APC-R700 (BD Bioscience), anti-CD45- BUV737 (BD Biosciences), and anti-CD11B-APC (Thermo Scientific). Fluorescence minus 1 was used as a negative control. Samples were collected on a Cytek Aurora flow cytometer (Cytek Biosciences). Analysis was performed using FlowJo software version 10.8.0. Microglia were identified as CD45 intermediate/CD11B high cell population among live cells.
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