BMDCs were collected after pulsed for 24 h and maturation markers expressed on BMDCs surface were analyzed by flow cytometry (FACS). The following monoclonal antibodies (mAb) were used: PerCP-Cyanine5.5-conjugated anti-CD11c, FITC-conjugated anti-CD80, PE-conjugated anti-CD86, and APC-conjugated anti-MHC class II (eBioscience, USA). The cells were respectively incubated with the mAb for 30 min at 4°C in the dark, and then washed twice with PBS containing 0.5% BSA and resuspended in PBS. FACS was performed on a Beckman Coulter Gallios cytometer and analyzed by using Kaluza software (Beckman Coulter, USA). To assess IL-10 and IL-12p70 levels produced by BMDCs, the culture supernatants were centrifuged and harvested at different time points (24 h, 36 h and 48 h) after stimulation and determined by ELISA using the corresponding mouse ELISA kits (eBioscience, USA) referred to the instructions.
Pe conjugated anti cd86
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE-conjugated anti-CD86 is a monoclonal antibody that binds to the CD86 antigen. CD86 is a costimulatory molecule expressed on the surface of antigen-presenting cells. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD86-expressing cells using flow cytometry.
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Lab products found in correlation
Fitc conjugated anti cd40, by Thermo Fisher Scientific (2 mentions)
Gallios cytometer, by Beckman Coulter (1 mentions)
Mouse elisa kit, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd80, by Thermo Fisher Scientific (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Mouse igg1 fitc, by BD (1 mentions)
Mouse igg2b, by Thermo Fisher Scientific (1 mentions)
Pe mouse igg1, by BD (1 mentions)
Navios, by Beckman Coulter (1 mentions)
Apc conjugated anti cd206, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Cyan adp cytometer, by Beckman Coulter (1 mentions)
Fc receptor block, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd11c, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Pe conjugated anti cd40, by Thermo Fisher Scientific (1 mentions)
6 protocols using pe conjugated anti cd86
1
Bone Marrow Dendritic Cell Maturation
2
Cell Surface Marker Analysis by Flow Cytometry
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For cell-surface molecular analysis, cells were suspended in PBS containing 1% FBS, and then stained with PE-conjugated anti-CD86 (12-0869-42), FITC-conjugated anti-CD206 (MA5-16870), PE-conjugated anti-CD163 (12-1639-42), Mouse IgG2b κ Isotype Control PE (12-4732-81), Mouse IgG1, κ Isotype Control Alexa Fluor 488 (53–4714) (all the antibodies purchased from eBioscience, USA), PE/cyanine 5-conjugated anti-CD11b (E-AB-F1081G, Elabscience, China), PE/cyanine 5 Rat IgG2b, κ Isotype Control (E-AB-F09842G, Elabscience, China), CD4-FITC/CD8-PE/CD3-PerCP (340298), CD3-FITC/CD16+56 PE (340042), Mouse IgG1 PE (349043), Mouse IgG1 FITC (349041), Mouse IgG2a PerCP (349054) (all the antibodies purchased from BD, USA) for 30 min at 4°C. For flow cytometry (FCM) gating, cells were stained with isotype-matched control antibodies or unstained cells and other cells were analyzed according to that fating strategy. Specimens were subsequently analyzed by FCM (Navios, Beckman).
3
Flow Cytometry for M1/M2 Macrophage Analysis
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Flow cytometry was performed for examining the surface marker expression on M2 macrophages. Briefly, the cells were stained with PE-conjugated anti-CD86 (Cat# 12-0869-42, eBiosciences, CA, USA) or APC-conjugated anti-CD206 (Cat# 17-2069-42, eBiosciences, CA, USA) in the dark at room temperature for 30 min. Subsequently, the cells were washed with PBS supplemented with 2% FBS, and the percentage of CD86+ cells—identified as M1 macrophages, the percentage of CD206+ cells—identified as M2 macrophages—were analysed by flow cytometer. The raw data was analysed by CytExpert software 2.3.
4
Microglial Surface Marker Modulation by TGF-β1
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To measure the effect of TGF-β1 on microglial surface marker expression after 3-AP infusion, flow cytometry was conducted. On day four after TGF-β1 injection, rats were anesthetized with sodium pentobarbital (55 mg/kg) and perfused with cold PBS via the left ventricle of the heart to eliminate cells in the vasculature. Brain stem and cerebellum tissues were isolated, respectively, and digested with collagenase D (2.5 mg/ml; Roche, Penzberg, Germany) and DNaseI (1 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) at 37 °C for 20 min. Then, the single cells were collected by passing the tissues through a 70 μm cell strainer, followed by a percoll gradient (25 and 75%) centrifugation. The mononuclear cells at the 25/75 interphase were obtained for subsequent analysis. The cells were labeled with APC-conjugated anti-CD11b, FITC-conjugated anti-CD40 or PE-conjugated anti-CD86 (all from eBioscience, San Diego, CA, United States) or the appropriate isotype control antibody. The frequency of the respective immunoreactive cells was expressed as a percentage in total mononuclear cells using a FACS Calibur flow cytometer (BD Biosciences, United States) equipped with CellQuest software.
5
Phenotypic Analysis of Bone Marrow Dendritic Cells
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Bone marrow-derived dendritic cells were stained in FACS buffer (PBS containing 0.1% bovine serum albumin and 5 mM EDTA) using the following monoclonal antibodies in the indicated concentration for cell surface markers (all obtained from eBioscience): PE-conjugated anti-CD86 (2 μg/ml), FITC conjugated anti-CD-40 (5 μg/ml), PE-conjugated anti-CD11c (1 μg/ml), and APC-conjugated MHC-II (0.28 μg/ml). Cells were first incubated with Fc receptor block, 5 μg/ml (eBioscience) for 10 min to block any non-specific binding and subsequent staining steps were performed for 20 min at 4°C, followed by washing with FACS buffer. An isotype-matched control staining was done for each antibody to determine a specific background staining. Cells were acquired using a Cyan-ADP cytometer (Beckman Coulter, Woerden, The Netherlands) and analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA).
6
Endogenous CD4 T cell responses and B cell costimulatory molecules in infected chimera
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Endogenous CD4 T cell responses in infected chimera were analyzed as described previously (Nothelfer et al., 2015) . Briefly, splenocytes were either restimulated with bone marrow-derived dendritic cells (BMDCs) pulsed with fixed parasites or phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of Brefeldin A (BD Biosciences). Cells were then stained with anti-CD4-fluorescein isothiocyanate (FITC), anti-CD8 Pacific Blue, anti-IFN-g-allophycocyanin, and anti-IL-10-phycoerythrin (BD Biosciences). 350,000 cells were acquired on a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
The expression of costimulatory molecules by B cells from infected chimeric mice was assessed using the following antibodies: FITC-conjugated anti-major histocompatibility complex class II (MHCII) (BD Biosciences), eFluor 450-conjugated anti-CD19 (eBioscience), PE-conjugated anti-CD40 (eBioscience), PE-conjugated anti-CD80 (eBioscience), and PE-conjugated anti-CD86 (eBioscience). Cells were acquired with a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
The expression of costimulatory molecules by B cells from infected chimeric mice was assessed using the following antibodies: FITC-conjugated anti-major histocompatibility complex class II (MHCII) (BD Biosciences), eFluor 450-conjugated anti-CD19 (eBioscience), PE-conjugated anti-CD40 (eBioscience), PE-conjugated anti-CD80 (eBioscience), and PE-conjugated anti-CD86 (eBioscience). Cells were acquired with a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
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