TIRF Microscopy for BCR Signaling Analysis

PLBs containing biotinylated anti-mouse Ig surrogate antigen were prepared following our published protocols7 (link)8 (link)15 (link)32 (link). B cells were firstly stained with Alexa Fluor 647 conjugated Fab anti-mouse IgG, Fc specific and then loaded to the chamber to react with surrogate antigens on the PLBs for 10 min followed by 4% paraformaldehyde (PFA) fixation. TIRFM images were captured by an Olympus IX-81 microscope supported by Andor iXon+ DU-897D electron-multiplying CCD camera, Olympus 100 × 1.49 NA objective lens and a TIRF port. TIRFM image acquisition was controlled by Metamorph software (Molecular Devices) and the exposure time for 512 × 512 pixels image was 100 ms. Total fluorescence intensity (TFI) of BCRs and BCR downstream signalling molecules accumulated to the IS were statistically analysed based on the intensity and area by Image J (NIH, USA)15 (link)32 (link).

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Chen X., Pan W., Sui Y., Li H., Shi X., Guo X., Qi H., Xu C, & Liu W. (2015). Acidic phospholipids govern the enhanced activation of IgG-B cell receptor. Nature Communications, 6, 8552.

Publication 2015

IX-81 microscope Metamorph software

Alexa fluor 647 Anti igg Antigens B cells Devices Electron Fluorescence Lens Microscope Mouse Paraformaldehyde

Corresponding Organization :

Other organizations : Tsinghua University, Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Center for Excellence in Molecular Cell Science, Center for Life Sciences

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Variable analysis

independent variables

Biotinylated anti-mouse Ig surrogate antigen on PLBs

dependent variables

Total fluorescence intensity (TFI) of BCRs and BCR downstream signalling molecules accumulated to the IS

control variables

Exposure time for 512 × 512 pixels TIRFM image (100 ms)
B cells stained with Alexa Fluor 647 conjugated Fab anti-mouse IgG, Fc specific

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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