Journal: iScience

Article Title: High-throughput single-cell antibody secretion quantification and enrichment using droplet microfluidics-based FRET assay

doi: 10.1016/j.isci.2022.104515

Figure Lengend Snippet: Single-cell antibody secretion assay in droplets using FRET (A) The schematics of the FRET-based assay. The assay mix containing cell growth medium, Alexa Fluor 488-labeled secondary antibody (FRET donor), Alexa Fluor 647-labeled c-myc peptide (FRET acceptor) are encapsulated in 40 pl droplets along with anti- c -myc antibody-secreting 9E10 hybridoma cells (blue). Following incubation off-chip at 37°C, the secreted and membrane-bound antibody fractions are recorded using FRET (emission from Alexa Fluor 647). In the presence of the antibody, the two labeled probe molecules form a ternary complex enabling the FRET reaction to occur. (B) Images of droplets acquired during 60 min of incubation using a widefield fluorescence microscope. The fluorescence intensity is color-coded, with red color pixels indicating the highest fluorescence intensity and blue the lowest. Scale bar, 50 μm. (C) FRET acceptor fluorescence intensity of droplets as a function of time. A clear increase in droplet fluorescence intensity (with a slope of 1.18 nM/min - red dashed line, and with a slope of 0.38 nM/min - blue dashed line) over time is observed, indicating the accumulation of the secreted antibody of interest. (D) The FRET acceptor fluorescence intensity of encapsulated cells. Cell fluorescence emanating from antibodies displayed at the cell membrane remains relatively stable over time. In panels C and D, the Y axes indicate the integrated FRET intensity subtracted by the average background FRET signal of droplets having no cells. Boxplots in C and D show the median (red lines) with upper and lower quartiles (blue lines), bars indicate the extremes of the distribution and crosses indicate outliers. See also and .

Article Snippet: The cell encapsulation process was monitored using a high-speed camera (Phantom Miro M310, Vision Research) mounted onto an inverted microscope (Nikon Ti-E, Nikon).

Techniques: Labeling, Incubation, Membrane, Fluorescence, Microscopy

Journal: iScience

Article Title: High-throughput single-cell antibody secretion quantification and enrichment using droplet microfluidics-based FRET assay

doi: 10.1016/j.isci.2022.104515

Figure Lengend Snippet:

Article Snippet: The cell encapsulation process was monitored using a high-speed camera (Phantom Miro M310, Vision Research) mounted onto an inverted microscope (Nikon Ti-E, Nikon).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Inverted Microscopy, Fluorescence, Hybridization