Journal: Scientific Reports

Article Title: High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

doi: 10.1038/s41598-019-47388-x

Figure Lengend Snippet: Functional cell-based assay for evaluating the reporter cell lines. ( a ) Functional cell-based assay using authentic exendin-4 (Ex4). hGLP1R/NLuc-293 and hGLP1R/LacZ-293 were cultured in DMEM media with or without 30 nM authentic Ex4. WT HEK293 was used as a control. Values are mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (* p < 0.05, ** p < 0.01). N.S., not significant. ( b ) Functional cell-based assay using culture supernatants of yeast cells secreting Ex4. hGLP1R/NLuc-293 and hGLP1R/LacZ-293 were cultured in DMEM media with culture supernatants of yeast cells producing Ex4 (Yeast-Ex4) or WT yeast cells (Yeast-WT). Values are mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (* p < 0.05, ** p < 0.01). N.S., not significant.

Article Snippet: To perform a functional assay using the adherent hGLP1R/LacZ-293 cell line, hGLP1R/LacZ-293 cells were seeded at 5 × 10 3 cells/100 µL in a 96-well plate (Thermo Fisher Scientific) and cultured at 37 °C for 24 h. The medium was then exchanged with 50 µL DMEM containing a hGLP1R ligand and 50 µM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific).

Techniques: Functional Assay, Cell Based Assay, Cell Culture, Two Tailed Test

Journal: Scientific Reports

Article Title: High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

doi: 10.1038/s41598-019-47388-x

Figure Lengend Snippet: Functional cell-based assay using floating reporter cells. ( a ) Functional cell-based assay using authentic Exendin-4 (Ex4). The floating hGLP1R/LacZ-293 cells were suspended in a RPMI-1640 medium with or without 30 nM of authentic Ex4. Floating WT HEK293 was used as control. Values are mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (* p < 0.05). N.S., not significant. ( b ) Functional cell-based assay by co-culture with yeast cells secreting Ex4. The floating mammalian hGLP1R/LacZ-293 or WT HEK293 cells were co-cultured with yeast cells producing Ex4 (Yeast-Ex4) or WT yeast cells (Yeast-WT). Values were mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (* p < 0.05). N.S., not significant.

Article Snippet: To perform a functional assay using the adherent hGLP1R/LacZ-293 cell line, hGLP1R/LacZ-293 cells were seeded at 5 × 10 3 cells/100 µL in a 96-well plate (Thermo Fisher Scientific) and cultured at 37 °C for 24 h. The medium was then exchanged with 50 µL DMEM containing a hGLP1R ligand and 50 µM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific).

Techniques: Functional Assay, Cell Based Assay, Two Tailed Test, Co-Culture Assay, Cell Culture

Journal: Scientific Reports

Article Title: High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

doi: 10.1038/s41598-019-47388-x

Figure Lengend Snippet: High-throughput functional cell-based assay using droplet microfluidics. ( a ) Representative fluorescence micrographs of droplets. The hGLP1R/LacZ-293 cells were encapsulated with no ligand, 30 nM authentic Exendin-4 (Ex4), WT yeast cells (Yeast-WT, 9.1 × 10 6 cells/mL), or yeast cells producing Ex4 (Yeast-Ex4, 9.1 × 10 6 cells/mL.). The images show the bright field images (upper image) and the fluorescence images (lower image). ( b ) Box plots of fluorescence intensity of each droplet in the experiment of ( a ). We analyzed at least 70 droplets containing a reporter cell for all samples. Statistical significance was determined by two-tailed Student’s t -test (** p < 0.01). N.S., not significant. ( c ) Box plots of fluorescence intensity of droplets. The hGLP1R/LacZ-293 cells were encapsulated with a mixture of yeast-WT cells and yeast-Ex4 cells at a ratio of 100:1. ( d ) A representative fluorescence image of droplets in the experiment of ( c ). The images show the bright field image (upper image) and the fluorescence image (lower image). ( e ) A colony-direct PCR was performed on yeast cells isolated in the experiment of ( c , d ). To determine whether colonies isolated from the strongly fluorescent droplet were yeast-WT or yeast-Ex4, a colony-direct PCR was performed on isolated cells. Yeast-Ex4 and yeast-WT were used as a positive control and a negative control, respectively.

Article Snippet: To perform a functional assay using the adherent hGLP1R/LacZ-293 cell line, hGLP1R/LacZ-293 cells were seeded at 5 × 10 3 cells/100 µL in a 96-well plate (Thermo Fisher Scientific) and cultured at 37 °C for 24 h. The medium was then exchanged with 50 µL DMEM containing a hGLP1R ligand and 50 µM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific).

Techniques: High Throughput Screening Assay, Functional Assay, Cell Based Assay, Fluorescence, Two Tailed Test, Isolation, Positive Control, Negative Control

Journal: Scientific Reports

Article Title: High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

doi: 10.1038/s41598-019-47388-x

Figure Lengend Snippet: Functional evaluation of yeast cells producing randomized Exendin-4 (Ex4) variants isolated by high-throughput functional cell-based assay using droplet microfluidics. ( a ) Evaluation of the activity of the randomized Ex4 variants secreted by isolated yeast cells. The adherent hGLP1R/NLuc-293 cells were incubated with culture supernatants of the isolated yeast cells, and luminescence intensity was quantified. Yeast-Ex4 and Yeast-WT were used as a positive control and a negative control, respectively. Values were given as mean ± SD (n = 3). ( b ) Sequences of the isolated Ex4 variants. Amino acids shown in red indicate the N-terminal two amino acids.

Article Snippet: To perform a functional assay using the adherent hGLP1R/LacZ-293 cell line, hGLP1R/LacZ-293 cells were seeded at 5 × 10 3 cells/100 µL in a 96-well plate (Thermo Fisher Scientific) and cultured at 37 °C for 24 h. The medium was then exchanged with 50 µL DMEM containing a hGLP1R ligand and 50 µM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific).

Techniques: Functional Assay, Isolation, High Throughput Screening Assay, Cell Based Assay, Activity Assay, Incubation, Positive Control, Negative Control

Journal: Scientific Reports

Article Title: High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics

doi: 10.1038/s41598-019-47388-x

Figure Lengend Snippet: Functional evaluation of Exendin-4 (Ex4) variants produced by E . coli. ( a ) The functional assay using WT Ex4 produced by E . coli . We cultured E . coli producing WT Ex4 fused with a FLAG sequence at the N-terminal ( E . coli -Ex4) and WT E . coli ( E . coli -WT). The cell lysates were purified using anti-FLAG resin, reacted with or without enterokinase, and assayed with the adherent hGLP1R/NLuc-293 cells. DMEM media with or without 3 nM authentic Ex4 were used as a positive control and a negative control. Values are mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (** p < 0.01). N.S., not significant. ( b ) Activities of each of the Ex4 variants produced by E . coli . Relative luminescence units were corrected based on concentrations of each peptide. Values are mean ± SD (n = 3). Two-tailed Student’s t -test was used to compare two groups (* p < 0.05, ** p < 0.01). N.S., not significant.

Article Snippet: To perform a functional assay using the adherent hGLP1R/LacZ-293 cell line, hGLP1R/LacZ-293 cells were seeded at 5 × 10 3 cells/100 µL in a 96-well plate (Thermo Fisher Scientific) and cultured at 37 °C for 24 h. The medium was then exchanged with 50 µL DMEM containing a hGLP1R ligand and 50 µM 5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG, Thermo Fisher Scientific).

Techniques: Functional Assay, Produced, Cell Culture, Sequencing, Purification, Positive Control, Negative Control, Two Tailed Test