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

Journal: Journal for Immunotherapy of Cancer

Article Title: Bivalent therapeutic vaccine against HPV16/18 genotypes consisting of a fusion protein between the extra domain A from human fibronectin and HPV16/18 E7 viral antigens

doi: 10.1136/jitc-2020-000704

Figure Lengend Snippet: In vivo induction of cellular immune responses against HPVE7 following immunization with EDA-HPVE7 vaccine candidates. (A) Schematic representation of the candidate fusion proteins. (B) Coomassie staining of the produced proteins (lane 1: mEDA-HPVE7-16; lane 2: molecular weight marker; lane 3: hEDA-HPVE7-16; lane 4: hEDA-HPVE7-16/18). (C) Binding of the fusion proteins to 293-toll-like receptor 4 (TLR4) and 293-LacZ expressing cells, measured by flow cytometry. (D) Tumor necrosis factor (TNF)-α production by THP-1 cells in response to stimulation with the indicated proteins. (E) Interleukin (IL)-12 production by human monocyte-derived dendritic cell (DC) in response to protein stimulation. (F) Number of interferon (IFN)-γ-producing cells specific for peptide E7 49–57 peptide in C57BL/6 mice previously immunized with the indicated protein±Poly IC. (G) Number of IFN-γ-producing cells specific for peptides from HPV16 and HPV18 genotypes in C57BL/6 mice previously immunized with hEDA-HPVE7-16/18 protein+Poly ICLC. (H) Antitumor efficacy of subcutaneous immunization with PBS, hEDA-HPVE7-16/18 alone or hEDA-HPVE7-16/18+Poly ICLC in C57BL/6 mice bearing TC-1 P3 (A15) tumors. Tumor size was measured at regular intervals. The ratio of tumor-free mice/total number of mice treated is shown at day 90 for each treatment. Survival following treatment is depicted using a Kaplan-Meier plot. Results are representative of two independent experiments. n.s., not significant; PBS, phosphate-buffered saline.

Article Snippet: To test whether the recombinant proteins were able to bind to TLR4 expressing cells, we used HEK293 expressing human TLR4-MD2-CD14 (HEK TLR4; Invivogen) or LacZ (HEK LacZ; InvivoGen) as a negative control.

Techniques: In Vivo, Staining, Produced, Molecular Weight, Marker, Binding Assay, Expressing, Flow Cytometry, Derivative Assay, Saline

Journal: bioRxiv

Article Title: Detecting Drug-Target Binding in Cells using Fluorescence Activated Cell Sorting Coupled with Mass Spectrometry Analysis

doi: 10.1101/121988

Figure Lengend Snippet: Expression of functional hKMO and compound screening in the TAPS assay. a) Transient transfection of HEK293 cells with E2-Crimson-huKMO generates populations of variably transfected cells, the histogram shows the distribution of fluorescence intensity in the transfected cells and the population gates applied for cell sorting, b) Cellular staining images obtained using the Opera™ High Content Screening system show variable expression of E2-Crimson in each sorted population. E2-Crimson-huKMO fluorescence was detected using 640 nm laser excitation (2000 μW, 280 ms), DAPI staining was detected using the UV light source (365 nm excitation, emission filter 450/50, 40 ms) and the 488 nm laser (1250 μW, 40 ms) was used for exciation the cell membrane stain (wheat germ agglutinin-Alexa488 conjugate). c) 3-HK produced per cell in each sorted cell population lysate after incubation with 200 μM kynurenine substrate for 2 hours correlates with fluorescence intensity in the cell.

Article Snippet: The source of KMO protein for this assay was lysate generated using the following stable cell line: HEK293 (Flp-In-293 which express lacZ-Zeocin, Life Technologies) cells were stably co-transfected with 9 μg of pcDNA5/FRT/V5/HisTOPO DNA and 18 μg of pog44 plasmid.

Techniques: Expressing, Functional Assay, Transfection, Fluorescence, FACS, Staining, High Content Screening, Membrane, Produced, Incubation