To measure cell membrane surface proteins, cells were collected after detachment with trypsin/EDTA, and immunostained for 30 min at 4 °C with fluorescence-conjugated primary antibodies. The following antibodies were used: PE-CD34, FITC-CD90, FITC-CD105, PE-MHC I, APC-MHC II (BD Biosciences Pharmingen, San Diego, CA, USA), and PE-HLA-G (MHC Ib) (Biolegend, CA, USA). After immunostaining, the cells were washed three times with PBS and suspended in 0.2 ml PBS for analysis. The fluorescence of samples was measured using a FACS Calibur (Becton Dickinson and Company, NJ, USA) and analyzed with FlowJo software (ver 10.6.1; Treestar, OR, USA).
Mhc 2 apc
Manufactured by BD
Sourced in United States
MHC-II-APC is a fluorescently-labeled antibody that binds to major histocompatibility complex class II (MHC-II) proteins. MHC-II proteins are expressed on the surface of antigen-presenting cells, such as dendritic cells, macrophages, and B cells, and play a crucial role in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (2 mentions)
Cd105 fitc, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
Cd34 pe, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Ccr3 pe, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
B220 fitc, by BioLegend (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cd206 pe, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Pharm lyse, by BD (1 mentions)
Cd83 pe, by BD (1 mentions)
Cd80 pe, by Thermo Fisher Scientific (1 mentions)
Cd54 pe, by BD (1 mentions)
Cd11c percp cy5, by BD (1 mentions)
Mouse anti human cd80 fitc, by BD (1 mentions)
Cd1a fitc, by BD (1 mentions)
5 protocols using mhc 2 apc
1
Cell Surface Protein Immunophenotyping
2
Evaluating Dendritic Cell Activation
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The upregulation of surface markers on the DCs were measured to determine the immunostimulatory effects of the nanoparticles. DCs were prepared and BSA loaded hybrid cationic CHL NPs particles were added in the same manner as 4.2.12, and incubated for 24 h. Control groups consisted of blank media (negative) and LPS (100 ng/mL) (positive). The cells were collected and washed as in 4.2.12 and stained with anti-mouse FITC-CD40, PE-CD86, and APC-MHC-II (BD, UK). The cells were washed and stained with 7AAD for 10 min before being measured using a BD Accuri C6 flow cytometer.
3
Flow Cytometric Enumeration of Eosinophils
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For detection of eosinophils, red blood cells were lysed using ammonium chloride lysis buffer (Sigma-Aldrich; Merck KGaA). Absolute counting beads (cat. no. C36950; Invitrogen; Thermo Fisher Scientific) were mixed with the BALF cells and assessed via flow cytometry. The numbers of BALF cells were calculated by comparing the ratio of beads events to cell events according to the manufacturer's protocol. The BALF cells were stained with antibodies against CD3-FITC (cat. no. 100203; Biolegend, Inc.), B220-FITC (cat. no. 103205, Biolegend, Inc.), CCR3-PE (cat. no. 144505; Biolegend, Inc.), CD11c-PerCP/Cyanine5.5 (cat. no. 117327; Biolegend, Inc.) and MHCII-APC (cat. no. 116417; BD Biosciences) for 30 min at 4°C. The cells were stained for neutrophils, mononuclear cells and lymphocytes (data not shown), and eosinophils using a CytoFLEX flow cytometer (Beckman Coulter, Inc.), and data collected were analyzed with FlowJo V10 software (FlowJo, LLC). Granulocytes were recognized as non-autofluorescent highly granular (SSChi) cells, and within this gate, eosinophils were defined as cells expressing the eotaxin receptor CCR3 and with low expression of MHCII, B220 and CD3. Neutrophils had a similar scatter profile as eosinophils but lacked CCR3 expression. Lymphocytes were identified as FSClo/SSClo and expressing CD3 or B220. Mononuclear cells expressed high levels of MHCII and CD11c.
4
Quantification of Macrophage Polarization
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Each group of abdominal aorta blood samples containing macrophages were washed and treated with BD Pharm Lyse (BD Biosciences, San Jose, CA, USA). Inactive cells were excluded by trypan blue. The live cells were incubated with MHC-II-APC (BD Biosciences, San Jose, CA, USA) and CD-206-PE (BD Biosciences, San Jose, CA, USA). The fluorescent stained cells were analyzed by a BD LSR II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). And the fluorescent compensation and data analysis were performed by FlowJo 7.5 software (TreeStar, Ashland, OR). MHCII+CD206- cells were indicated the M1 macrophages while MHCII+CD206+ cells were M2 macrophages. This assay was repeated at least three times.
5
Flow Cytometric Analysis of Dendritic Cells
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Isolated CD103+ DCs (1 × 106) were subjected to flow cytometric analysis. Mouse-anti-rat CD54-Alexa Fluor®488 (R&D Systems), CD86-phycoerythrin (PE)-Vio770 (Miltenyi Biotec), CD80-PE (eBioscience, San Diego, CA, USA), CD11b/c-allophycocyanin (APC; Biolegend, San Diego, CA, USA), RT1B (MHC- II)-FITC (BD Biosciences, San Jose, CA, USA), CD1d-APC (eBioscience), and the respective matched isotype controls were employed.
The following markers were used for the staining of imDCs (1 × 106): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).
The following markers were used for the staining of imDCs (1 × 106): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).
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