FRET measurement

Note: This assay is applied to determine extracellular orientation change of transmembrane proteins on cell plasma membrane. Here, we use FcγRIIB, an immune receptor, for example. This protocol has been adapted from Hu et al., eLife, 2019.

Materials and Reagents
mTFP cDNA, pHAGE vector, psPAX2 vector, pMD2.G vector, A20II1.6 B cell lines (ATCC), ClonExpress™ MultiS One Step Cloning Kit (Catalog#C113, Vazyme, China), Octadecyl rhodamine B (R18) (Catalog#O246, Invitrogen), Nunc Glass Bottom Dishes (Catalog#150680, ThermoFisher).

Equipment
MoFlo Astrios EQ, Cell Sorter (Beckman) and TiE C2 confocal microscope (Nikon)

Software
GraphPad Prism and Image J

Procedure
Step One: Plasmid construction and cell lines establishment
1.1 mTFP cDNA was fused to the N termini of FcγRIIB in a pHAGE backbone by ClonExpress™ MultiS One Step Cloning Kit.
1.2 mTFP-FcγRIIB expressing A20II1.6 B cell lines were acquired by lentivirus infection (three-vector system: mTFP-FcγRIIB pHAGE, psPAX2, and pMD2.G).
1.3 A20II1.6 B cell line stably expressing mTFP-FcγRIIB with similar expression level was obtained by multiple rounds of cell sorting (Obtaining similar expression levels different experimental groups is very important if you need to accurately compare different transmembrane proteins’ orientation on cell plasma membrane, as different expression level may affect FRET ratio).

Step Two: FRET measurement
2.1 1×10⁶ mTFP-FcγRIIB A20II1.6 B cells were collected and washed at 4℃ and 1200 rpm for 5 min once with 1×PBS.
2.2 mTFP-FcγRIIB A20II1.6 B cells were re-suspended with 1 mL cell buffers (Note: select different cell buffers according to the experiment purpose. Here we use the cell buffer containing HEPES 10 mM, KH₂PO₄ 1.3 mM, NaCl 130 mM, CaCl₂ 1.5 mM, MgSO₄ 0.5 mM, Dextrose 34 mM, pH=7.35 and 275-290 mOsm osmolality).
2.3 mTFP-FcγRIIB A20II1.6 B cells were stained with 300 nM octadecyl rhodamine B (R18) on ice for 3 min.
2.4 Centrifuge and washed at 4℃ and 1200 rpm for 5 min twice with cell buffers.
2.5 The above cells were added into Nunc Glass Bottom Dishes and then mounted onto a Nikon TiE C2 confocal microscope with 100x oil lens and maintained for 5 min to allow cells to settle down to the glass bottom.
2.6 Determine mTFP (457 nm laser; 515/30 bandpass filter) fluorescence intensity before and after R18 (561 nm laser; 595/50 bandpass filter) quenching.
2.7 FRET efficiency was calculated with the formula FRET efficiency=(DQ-Q)/DQ, DQ and Q represented dequenched and quenched mTFP fluorescence intensity respectively. Note: mTFP fluorescence intensity was measured by Image J.
 

References
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 6:1–14.
Hu W, Zhang Y, Sun X, Zhang T, Xu L, Xie H, Li Z, Liu W, Lou J, Chen W. FcγRIIB-I232T polymorphic change allosterically suppresses ligand binding. eLife, 2019, 8: e46689
Xu C, Gagnon E, Call ME, Schnell JR, Schwieters CD, Carman CV, Chou JJ, Wucherpfennig KW. 2008. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif. Cell 135:702–713.

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