Tissue samples were embedded in optimal cutting temperature compound (OCT) and snap frozen in liquid nitrogen. Tissue sections (4 μm) were fixed in ethanol/acetic acid fixative solution for 2–10 min, and then were stained with anti-F4/80-PE (eBioscience), CD206-PerCP-Cy5.5 (Biolegend) overnight at 4°C in a humidified chamber. After three times of washes, slides were stained with DAPI and mounted with Prolong Gold mounting reagent (Invitrogen). Confocal imaging was performed using Leica SP5 II confocal microscope.
Cd206 percp cy5
Manufactured by BioLegend
CD206-PerCP-Cy5.5 is a fluorescently labeled antibody that binds to the CD206 receptor, also known as the mannose receptor. CD206 is a cell surface receptor expressed on the surface of macrophages and dendritic cells. The PerCP-Cy5.5 fluorescent label allows for the detection and analysis of CD206-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti f4 80 pe, by Thermo Fisher Scientific (1 mentions)
Sp5 2 confocal microscope, by Leica (1 mentions)
Prolong gold mounting reagent, by Thermo Fisher Scientific (1 mentions)
Gr 1 pecy7, by Thermo Fisher Scientific (1 mentions)
Anti mouse f4 80 fitc, by Thermo Fisher Scientific (1 mentions)
Cd86 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd11c apccy7, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
F4 80 pe cy7 bm8, by BioLegend (1 mentions)
Fortessa, by BD (1 mentions)
Cd11b bv785, by BioLegend (1 mentions)
Cd64 af647, by BioLegend (1 mentions)
Cd45 bv605, by BioLegend (1 mentions)
Mhcii af700, by BioLegend (1 mentions)
Cd45 pacific blue, by BioLegend (1 mentions)
Cd8 pe, by BioLegend (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Tigit pe cy7, by BioLegend (1 mentions)
Foxp3 alexa488, by BioLegend (1 mentions)
6 protocols using cd206 percp cy5
1
Immunostaining of Macrophage Subsets
2
Characterizing Melittin Binding to Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rhodamine-conjugated melittin peptides were purchased from GenScript (Piscataway, NJ, USA). Splenocytes were seeded in a 6-well culture plate with 0.5 μg/ml of rhodamine-conjugated melittin. An hour later, cells were harvested and unbound peptides were washed off twice. Cells were stained with APC-conjugated antibodies for an hour at 4 °C to confirm the binding of melittin to CD4+ and CD8+ T cells and CD11b+ monocytes.
To verify whether the binding of melittin to CD11b+ cells is related to phagocytosis, splenocytes were pretreated with 10 nM cytochalasin D or vehicle (DMSO) for an hour in a 37 °C incubator. Next, the cells were incubated with the rhodamine-conjugated peptides and stained with CD11b-APC antibody as described above.
To observe the binding of melittin on CD11b+ subsets in splenocytes, macrophage, dendritic cells, and neutrophils were probed with anti-mouse F4/80-FITC, CD11c-APCcy7, and Gr1-PEcy7 (e-bioscience). Annexin-V was added to the samples prior to data acquisition to discriminate dead cells. M1 of M2 macrophages were stained with CD86-PEcy7 (e-bioscience) or CD206-PercpCy5.5 (Biolegend). Cells were detected on a FACSCalibur or FACSCantoII.
To verify whether the binding of melittin to CD11b+ cells is related to phagocytosis, splenocytes were pretreated with 10 nM cytochalasin D or vehicle (DMSO) for an hour in a 37 °C incubator. Next, the cells were incubated with the rhodamine-conjugated peptides and stained with CD11b-APC antibody as described above.
To observe the binding of melittin on CD11b+ subsets in splenocytes, macrophage, dendritic cells, and neutrophils were probed with anti-mouse F4/80-FITC, CD11c-APCcy7, and Gr1-PEcy7 (e-bioscience). Annexin-V was added to the samples prior to data acquisition to discriminate dead cells. M1 of M2 macrophages were stained with CD86-PEcy7 (e-bioscience) or CD206-PercpCy5.5 (Biolegend). Cells were detected on a FACSCalibur or FACSCantoII.
3
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were obtained from BioLegend and used at a dilution of 1:200: CD45 BV605 (30‐F11), CD11b BV785 (M1/70), F4/80 PE‐Cy7 (BM8), SiglecF BV421 (S17007L), Ly6C APC‐Fire 750 (HK1·4), CD11c BV650 (N418), MHCII AF700 (M5/114·15·2), CD64 AF647 (X54‐5/7·1), MerTK FITC (2B10C42) and CD206 PerCP‐Cy5·5 (C068C2) for 30 min at 4°C. Ly6G BUV395 (1A8) and CD103 BUV805 (M290) were obtained from BD Bioscience. Dead cells were excluded using Fixable Viability Dye eFluor 506 (Thermo Fisher). Flow cytometry was performed using a Fortessa (BDBiosciences) and analysed using FlowJo V10.
4
Tumor Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were harvested, weighed, and processed to obtain single-cell suspensions. In brief, tumor tissues were dissociated and digested with 250 µg/mL of Liberase (Roche) and incubated for 30 min at 37 °C, while shaking at 105 rpm for proper digestion. Fetal bovine serum was then added to stop the reaction, and samples were filtered and washed with PBS + 2% FBS. The cell count per sample was performed, then samples were stained using fluorochrome-conjugated antibodies from BioLegend including: CD45 Pacific blue, cat# 103126; CD4 BV605, cat# 100451; CD8 PE, cat# 100707; CD49b APC, cat# 108910; Foxp3 Alexa488, cat# 126406; TIGIT PE-Cy7, cat# 142108; Gr1 BV510, cat# 108437; CD11b APC-fire750, cat# 101262; F4/80 Alexa700, cat# 123130; CD206 PercpCy5.5, cat# 141716. In alternate panels, a set of other antibodies was also used including: CD4 FITC, cat# 100406; CD8 PErcpCy5.5, cat# 100734; Gr1 APC, cat# 108412; CD11c BV510, cat# 117337; and PVR (CD155) PE, cat# 132206. Samples were run on the Attune flow cytometer, and data were analyzed using Flow-Jo 10 software.
5
Polarization of THP1 and Mouse Myeloid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP1 cells were pretreated with 50 ng/mL PMA (Sigma) for 48 hours, then stimulated with macrophage M2 polarization inducers IL4 and IL13 (both at 40 ng/mL, Peprotech) for 48 hours. THP1 or whole blood cells from mice were resuspended in Stain Buffer (BD, 554656) for 15 minutes. The THP1 cells were stained with CD11b-APC (BD, 550019), CD64-FITC (BD, 555527), CD86-FITC (BD, 557343), and CD206-FITC (BD, 551135). The whole blood cells were stained with Ly6C-PE/Cy7 (BioLegend, 128017), Ly6G-APC (BioLegend, 127613), CD11b-FITC (BioLegend, 101205), CD86-PE (BioLegend, 105106), and CD206-PerCP/Cy5.5 (BioLegend, 141716). The cells were detected on a CytoFLEX (Beckman Coulter). The results were analyzed by FlowJo v10.0.
6
Immune Cell Profiling in Irradiated Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Female C57BL/6J mice aged 12 weeks were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France). The mice were immobilised through anaesthesia (2% isoflurane) and locally irradiated at the thorax using a Varian Tube NDI 226 (X-ray machine; 250 Kev, tube current: 15 mA, beam filter: 0.2 mm Cu), with a dose rate of 1.08 Gy•min -1 . A single dose of 16 Gy was locally administered to the whole thorax.
Flow cytometry and fluorescence-activated cell sorting F4/80-FITC (eBioscience, Paris, France), Gr1-PE (eBioscience), CD11c-PE-Cy7 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), and CD11b-APC-Cy7 (Biolegend) were used to identify neutrophils (Gr1 high (Ly6G + Ly6C -)), monocytes (Gr1 + (Ly6G -Ly6C + )), AMs (CD11c + CD11b -F4/80 + ) [14] and IMs (Gr1 -CD11b + CD11c -/+ F4/80 + ) [15] . Gr1 -mononuclear phagocytes (MPs) were identified as Gr1 - CD11b + CD11c + F4/80 -. Icam1-Pacific blue (Biolegend) was used to identify M1-polarised macrophages and CD206-PerCpCy5.5 (Biolegend), to identify M2-polarised macrophages. CD115-PE (eBioscience) was used to examine CSF1R expression in AMs and IMs. The samples were analysed using FlowJo 10.0.7 (FlowJo, Ashland, OR, USA). IMs and AMs were sorted using fluorescence-activated cell sorting and transferred in the culture medium.
Flow cytometry and fluorescence-activated cell sorting F4/80-FITC (eBioscience, Paris, France), Gr1-PE (eBioscience), CD11c-PE-Cy7 (Biolegend, Ozyme, Montigny-le-Bretonneux, France), and CD11b-APC-Cy7 (Biolegend) were used to identify neutrophils (Gr1 high (Ly6G + Ly6C -)), monocytes (Gr1 + (Ly6G -Ly6C + )), AMs (CD11c + CD11b -F4/80 + ) [14] and IMs (Gr1 -CD11b + CD11c -/+ F4/80 + ) [15] . Gr1 -mononuclear phagocytes (MPs) were identified as Gr1 - CD11b + CD11c + F4/80 -. Icam1-Pacific blue (Biolegend) was used to identify M1-polarised macrophages and CD206-PerCpCy5.5 (Biolegend), to identify M2-polarised macrophages. CD115-PE (eBioscience) was used to examine CSF1R expression in AMs and IMs. The samples were analysed using FlowJo 10.0.7 (FlowJo, Ashland, OR, USA). IMs and AMs were sorted using fluorescence-activated cell sorting and transferred in the culture medium.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!