For internalisation assays, purified B cells were loaded with biotinylated anti-IgM (SouthernBiotech) on ice for 20 min. Excess antibody was then removed, and the cells were incubated at 37°C for the time specified and fixed using 4% formaldehyde. Antibody remaining on the surface of the cells was labelled using fluorescent streptavidin (eBioscience) and analysed using flow cytometry. For antigen presentation assay, B cells were loaded with microspheres decorated by biotinylated anti-IgM (SouthernBiotech) and Eα peptide for 30 min at 37°C. The cells were then washed to remove excess microspheres and were incubated at 37°C for the time specified. The cells were fixed using 4% formaldehyde and stained with anti-MHC-II:Eα antibody (eBioY-Ae) (eBioscience), followed by anti-mouse IgG2b antibody (Life Technologies) staining for detection by flow cytometry. To measure microspheres uptake, the cells were incubated with coated microspheres for 30 min on ice. The cells were washed and incubated at 37°C for up to 1 h. The cells were fixed at specific time points and labelled with fluorescently labelled streptavidin and analysed using flow cytometry.
Ebioy ae
Manufactured by Thermo Fisher Scientific
The EBioY-Ae is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform specific functions within a laboratory setting. Without further interpretation, this is the extent of the factual and unbiased description that can be provided for this product.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 450 streptavidin, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg2b antibody staining, by Thermo Fisher Scientific (2 mentions)
Soluble biotinylated anti igm, by Southern Biotech (2 mentions)
Biotinylated anti igm, by Southern Biotech (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Rat igg2b eb149 10h5, by Thermo Fisher Scientific (1 mentions)
Treg permeabilization buffers, by Thermo Fisher Scientific (1 mentions)
Clip 15g4, by Santa Cruz Biotechnology (1 mentions)
Rmp1 30, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
6 protocols using ebioy ae
1
B Cell Internalization and Antigen Presentation Assays
2
Multicolor Flow Cytometry Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used include: Ii (In-1, 1:100), H2-M (2E5A, 1:50), Rat IgG1 (R3-34, 1:50; all from BD Biosciences); podoplanin (8.1.1, BioLegend, 1:500); CLIP (15G4, Santa Cruz, 1:5); H2-O (ref. 69 (link); Mags.Ob3, Lisa Denzin, 1:400); 10.1.1 (UVA lymphocyte culture centre, 1:1,000); CD45 (30-F11, 1:1,000), CD31 (390, 1:1,000), MHC-II (M5/114.15.2, 1:50 (Fig. 1 ) to 1:500 (Fig. 6 )), CD8 (53-6.7, 1:1,000), CD4 (GK1.5, 1:1,000), Thy1.1 (HIS51, 1:1,000), Thy1.2 (53-2.1, 1:1,000), CD45.1 (A20, 1:500), CD11c (N418, 1:500), CD11b (M1/70, 1:500), CD69 (H1.2F3, 1:500), CD62L (MEL-14, 1:1,000), CD44 (IM7, 1:1,000), CD25 (PC61.5, 1:500), Y-AE (eBioY-Ae, 1:200), PD-1 (RMP1-30, 1:100), LAG-3 (eBioC9B7W, 1:100), Rat IgG2b (eB149/10H5; all from eBioscience). Intracellular staining for PD-1, LAG-3, Ii, H2-M and H2-O was done using the Cytofix/Cytoperm kit (BD Bioscience), and Ki67 (SolA15, 1:500) and FoxP3 (FJK-16s, 1:100) were stained using Treg permeabilization buffers (eBioscience). Annexin V (1:20) was stained using the eBioscience kit. 4,6-Diamidino-2-phenylindole (DAPI; Sigma) or live/dead aqua (Invitrogen, 1:200) were used to distinguish live cells. Cells were acquired on a FACSCanto II (BD Biosciences) and data analysed using FlowJo (Tree Star).
3
B Cell Antigen Internalization and Presentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For internalization assays, purified B cells were loaded with IgM coated beads or with soluble biotinylated anti-IgM (Southern Biotech) on ice for 30 min. Cells were then washed with PBS 2% FCS to remove excess antigen, and then incubated for 5–30 min at 37°C. After fixation with 2% paraformaldehyde, noninternalized beads were detected with Alexa Fluor 450 streptavidin (eBioscience).
To detect antigen presentation, B cells loaded with Ea peptide and IgM-coated beads were incubated between 3 and 5 h at 37°C, and then fixed in 2% formaldehyde. These cells were then stained with anti-MHCII/Ea antibody (eBioY-Ae; eBioscience), followed by anti–mouse IgG2b antibody staining (Life Technologies) for detection.
To detect antigen presentation, B cells loaded with Ea peptide and IgM-coated beads were incubated between 3 and 5 h at 37°C, and then fixed in 2% formaldehyde. These cells were then stained with anti-MHCII/Ea antibody (eBioY-Ae; eBioscience), followed by anti–mouse IgG2b antibody staining (Life Technologies) for detection.
4
Internalization and Antigen Presentation Assay for B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For internalization assays, purified B cells were loaded with IgM coated fluorescently-labelled microspheres or with soluble biotinylated anti-IgM (Southern Biotech) on ice for 30 min. Cells were then washed with PBS 2% FCS to remove excess antigen and then incubated for 5 to 30 min at 37°C. After fixation with 2% paraformaldehyde, non-internalised beads or anti-IgM were detected with AlexaFluor450 streptavidin (eBioscience, San Diego, California). In case of bead internalisation, bead-positive cells were selected on the basis of the bead fluorescence.
To detect antigen presentation, B cells loaded with Eα peptide and IgM coated microspheres were incubated between 3 and 5 hr at 37°C and then fixed in 2% formaldehyde. These cells were then stained with anti-MHCII/Eα antibody (eBioY-Ae, eBioscience), followed by anti-mouse IgG2b antibody staining (Life Technologies) for detection.
To detect antigen presentation, B cells loaded with Eα peptide and IgM coated microspheres were incubated between 3 and 5 hr at 37°C and then fixed in 2% formaldehyde. These cells were then stained with anti-MHCII/Eα antibody (eBioY-Ae, eBioscience), followed by anti-mouse IgG2b antibody staining (Life Technologies) for detection.
5
Detecting Antigen Presentation In Vitro and In Vivo
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect antigen presentation in vitro, B cells were incubated with microbeads (Bangs laboratories) conjugated to anti‐Igκ and Ea peptide (Biotin‐GSGFAKFASFEAQGALANIAVDKA‐COOH) for 5 h at 37°C, fixed in 4% PFA, and blocked with 2% BSA and Fc block.
For in vivo assays, mice were immunized (intraperitoneal) with SRBC‐HEL conjugated to Ea peptide, as previously described (Brink et al,2015 (link)). Splenocytes were harvested 24 h later and blocked with 2% BSA and Fc block on ice. For both assays, samples were then stained with antibody against Ea52–68 bound to I‐Ab (eBioY‐ae, eBioscience) at 1/100 dilution in PBS‐BSA.
For in vivo assays, mice were immunized (intraperitoneal) with SRBC‐HEL conjugated to Ea peptide, as previously described (Brink et al,2015 (link)). Splenocytes were harvested 24 h later and blocked with 2% BSA and Fc block on ice. For both assays, samples were then stained with antibody against Ea52–68 bound to I‐Ab (eBioY‐ae, eBioscience) at 1/100 dilution in PBS‐BSA.
6
BALB/c T and B Cell Co-culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BALB/c T and B cell purified by negative selection following the instruction provided by BioLegend and Stem Cells, respectively, were co-cultured with MutuDC1s or MutuDC2s for 3 hours in the absence or presence of standard dose of PolyG/EDTA. After incubation the cells were washed, stained with eBioscience’s Fixable Viability Dye eFluor780, I-A/I-E (M5/114.15.2) and YAe (eBioY-Ae) antibodies and analyzed by flow cytometer.
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!