PLBs containing biotinylated anti-mouse Ig surrogate antigen were prepared following our published protocols (40 (link)). B cells were first stained with Alexa Fluor 647 AffiniPure Fab fragment goat anti-mouse IgM, μ chain–specific (115-607-020, Jackson ImmunoResearch Laboratory) and then loaded on the chamber to react with surrogate antigens on the PLBs for at least 10 min, followed by 4% paraformaldehyde (PFA) fixation. TIRFM images were captured using an Olympus IX-81 microscope supported by an Andor iXon+ DU-897D electron-multiplying charge-coupled device camera, Olympus 100 × 1.49 numerical aperture objective lens, and TIRF port. TIRFM image acquisition was controlled by MetaMorph software (Molecular Devices), and the exposure time for 512 × 512 pixel images was 100 ms. The mFIs of BCR, FcRL1, and intracellular signaling molecules accumulated at the immunological synapse were statistically analyzed according to the intensity and area using ImageJ software [National Institutes of Health (NIH)].
Protocol detail
Quantifying B Cell Receptor Signaling
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Alexa fluor 647
Anti igm
Antigens
B cells
Devices
Electron
Fab fragment
Fcrl1
Goat
Immunological synapse
Intracellular
Lens
Microscope
Mouse
Paraformaldehyde
Corresponding Organization : Sungkyunkwan University
Other organizations : Lahore University of Management Sciences, Chengdu Institute of Biology, University of California, San Francisco, National Institutes of Health, National Cancer Institute
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Variable analysis
independent variables
- Biotinylated anti-mouse Ig surrogate antigen on PLBs
- Accumulation of BCR, FcRL1, and intracellular signaling molecules at the immunological synapse
- Intensity and area of accumulation of BCR, FcRL1, and intracellular signaling molecules
- Alexa Fluor 647 AffiniPure Fab fragment goat anti-mouse IgM, μ chain–specific for staining B cells
- Reaction time of B cells with surrogate antigens on PLBs (at least 10 min)
- 4% paraformaldehyde (PFA) fixation
- TIRFM imaging parameters (Olympus IX-81 microscope, Andor iXon+ DU-897D camera, Olympus 100 × 1.49 numerical aperture objective lens, 100 ms exposure time, 512 × 512 pixel images)
- Not explicitly mentioned
- Not explicitly mentioned
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