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: Evaluation of FRET-based antibody binding signals (A) Comparison of the fluorescence emission spectra associated with positive, negative, and reference samples upon excitation at 488 nm. The positive sample comprises Alexa Fluor 488-labeled secondary antibody (FRET donor), Alexa Fluor 647-labeled c-myc peptide (FRET acceptor), and anti- c -myc antibody. These components form a ternary complex, bringing the donor and acceptor molecules into closeness, proximity. This results in FRET excitation of the Alexa Fluor 647 dye, and an emission peak at 668nm. The negative and reference samples contain the same FRET donor/acceptor pair and either a negative control primary antibody (directed against IFN-γ) or no primary antibody, respectively. These samples display no discernible emission of Alexa Fluor 647. Hence, the FRET reaction only occurs in the presence of the antigen-specific antibody. CPS – counts per second. (B) Schematics of droplets containing different combinations of assay reagents.

Article Snippet: After loading the samples, the imaging chambers were mounted on an inverted microscope equipped with a fluorescence lamp (Lumencor Inc.) and camera (PCO AG), and imaged using a 20X objective (Nikon).

Techniques: Binding Assay, Comparison, Fluorescence, Labeling, Negative Control

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: After loading the samples, the imaging chambers were mounted on an inverted microscope equipped with a fluorescence lamp (Lumencor Inc.) and camera (PCO AG), and imaged using a 20X objective (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: Sorting of FRET-positive droplets to isolate cells secreting target-reactive antibodies (A) The schematics of the cell sorting based on antibody secretion. The assay mix containing cell growth medium, Alexa Fluor 488-labeled secondary antibody (FRET donor), and Alexa Fluor 647-labeled c-myc peptide (FRET acceptor) is encapsulated together with a mixture of hybridoma cells. The anti- c -myc antibody-secreting 9E10 cells are marked in blue, and the non-specific, anti-IFN-γ antibody-secreting 7R2/A4 cells are marked in dark red. The collected droplets are incubated off-chip at 37°C to induce antibody secretion and then re-introduced into the microfluidic droplet-sorting device for sorting based on the red FRET acceptor fluorescence signal (emission from Alexa Fluor 647). In the absence of the antigen-specific antibody, the acceptor molecule is not bound and therefore not excited upon illumination with the 488 nm laser. Thus, only emission from the green FRET donor is observed. In the presence of the desired antibody (blue), the two labeled probe molecules form a ternary complex and the FRET donor and acceptor molecules are brought in closeness, proximity, enabling the FRET reaction to occur. In this case, in addition to the green FRET donor emission, red light is emitted by the FRET acceptor molecule. (B) Digital micrographs of droplets before and after FRET-based sorting. Before sorting, the desired cells (red) are rare, such that no strong FRET signal is observed with the majority of encapsulated cells displaying the Hoechst 33,342 stain (blue), identifying these cells as undesired 7R2/A4 cells. After sorting, a significant enrichment of desired cells is observed. The discarded fraction mostly consists of droplets containing the undesired cell type, or no cell. Scale bars, 50 μm. See also and .

Article Snippet: After loading the samples, the imaging chambers were mounted on an inverted microscope equipped with a fluorescence lamp (Lumencor Inc.) and camera (PCO AG), and imaged using a 20X objective (Nikon).

Techniques: FACS, Labeling, Incubation, Fluorescence, Staining

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: Time traces and scatterplot of FRET signal during microfluidic sorting (A) Relative maximum donor/acceptor fluorescence signals recorded during the sorting process. The red dashed line represents the chosen sorting threshold, with sorted droplets indicated by red dots. Color bar indicates droplet number in log scale. (B–E) Representative fluorescence time traces recorded throughout the sorting experiment. Both FRET donor fluorescence (green) and FRET acceptor fluorescence (red) are recorded. All droplets show strong donor fluorescence, allowing direct detection of each droplet. The FRET acceptor fluorescence intensity originating from positive droplets depends on Ab expression by individual cells. Droplets containing cells that secrete a large amount of target-reactive Ab display an elevated fluorescence pedestal (B), while those bearing cells with primarily membrane-bound target-reactive Ab are characterized by a narrow fluorescence peak (C). Cells that produce both secreted and membrane-bound forms of target-reactive Ab are characterized by an elevated fluorescence pedestal with a narrow fluorescence peak (D). Empty droplets and those containing cells that secrete an irrelevant Ab display low fluorescence in the red channel (E). (F–I) Selected examples of color-coded droplets containing: a cell secreting large quantities of target-reactive Ab and producing only a small amount of membrane-associated Ab (F); a cell producing high levels of membrane-bound Ab (G); a cell producing high quantities of both, secreted and membrane-bound Ab fractions (H); and a cell showing reduced levels of Ab expression and/or cell producing unreactive (non-functional) Ab (I).

Article Snippet: After loading the samples, the imaging chambers were mounted on an inverted microscope equipped with a fluorescence lamp (Lumencor Inc.) and camera (PCO AG), and imaged using a 20X objective (Nikon).

Techniques: Fluorescence, Expressing, Membrane, Functional Assay

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: After loading the samples, the imaging chambers were mounted on an inverted microscope equipped with a fluorescence lamp (Lumencor Inc.) and camera (PCO AG), and imaged using a 20X objective (Nikon).

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