Journal: Nature biotechnology

Article Title: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

doi: 10.1038/s41587-020-0466-7

Figure Lengend Snippet: Fig. 1 | The CelliGO process. a, Schematic of sorting on the basis of IgG binding to the soluble antigens TT and GPI (top panel) or to the membrane antigen TSPAN8 (middle panel), followed by paired VH–VL sequencing of the sorted cells (bottom panel). See text for details. b, Images of microfluidic operations. From left to right: droplet production, droplet sorting and co-compartmentalization of single cells with single beads in droplets (see also Supplementary Movie). Scale bars, left and middle 100 µm, right 40 µm. c, CelliGO timeline. The total time for the entire process from cell harvesting to antibody validation takes 20 d, including only 12 h from cell harvesting to NGS. PMTs, photomultiplier tubes; RT, reverse transcription.

Article Snippet: Affinity was determined as follows: anti-TSPAN8 IgG antibodies that demonstrated specific binding to TSPAN8+ M300.19 cells were fluorescently labeled with Alexa Fluor 488 and incubated in serial dilutions with TSPAN8+ M300.19 cells in ice-cold FACS buffer (Ca2+, Mg2+-free PBS supplemented with 10% FBS) for 30 min on ice, and cell-bound fluorescence was quantified using a MACSQuant flow cytometer (Miltenyi Biotech).

Techniques: Binding Assay, Membrane, Sequencing, Cell Harvesting, Biomarker Discovery, Reverse Transcription

Journal: Nature biotechnology

Article Title: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

doi: 10.1038/s41587-020-0466-7

Figure Lengend Snippet: Fig. 3 | Sequencing, expression and characterization of IgGs expressed by sorted cells. a, In-droplet single-cell barcoding of VH and VL cDNA. Cells from sorted droplets were recovered and re-compartmentalized individually in new droplets in the presence of reverse transcription reagents and a hydrogel bead bearing ~109 copies of VH–VL RT primers tagged with a bead-specific DNA barcode, which was released from the hydrogel beads by ultraviolet photocleavage. After breaking the emulsion, the pooled, barcoded VH–VL cDNAs were amplified and processed for NGS. b,c, Diversity of V-gene families use in paired VH–VL sequences from the TT-immunized mice (TT1 + TT2) (b) and the TSPAN8-immunized mice (TSPAN8-1 + TSPAN8-2) (c). Phylogenies were created using the full-length amino acid sequences of the V regions. Sequences are color-coded by the closest germline V-gene family. See also Supplementary Figs. 13–15. d, Cross-phylogeny of ten unique paired VH–VL sequences from the GPI2 sort derived from the same recombined germline V(D) J genes, illustrating co-evolution of VH and VL during affinity maturation. One candidate from each cluster (indicated by (1), (2) and (3) on the right side of tree) was expressed and was tested for binding to GPI (shaded yellow boxes); the three IgGs had similar affinity (Kd = 0.195–0.375 nM). e, The sequences of the complementarity determining regions (CDRs) from the paired VH–VL sequences presented in panel d. One candidate from each cluster was expressed and was tested for binding to GPI (shaded yellow boxes); the three IgGs had similar affinity (Kd = 0.195–0.375 nM). f, Histogram of frequency (percentage) of paired VH–VL sequences versus clonotype size. The clonotype size is equal to the number of non-redundant VH–VL sequences derived from the same ancestral VH–VL germline recombination event (that is, derived from the same germline V-J genes, with CDR3s of the same length and differing by at least one amino acid across the variable region). Inset: Schematic of IgG clustering on the basis of clonal expansion and somatic hypermutation of a single ancestral cell. Germline VH and VL genes are indicated by red and blue rectangles, and vertical white bars indicate mutations. Dark blue regions define clusters of clones (VH–VL pairs with exactly the same amino acid sequence but associated with different barcodes and therefore derived from different cells) (size = 2, 2 and 1). The number of dark blue regions in a light blue region defines the clonotype size (size = 3). See also Supplementary Table 4. g, Affinities of purified anti-TT IgGs (EC50 by ELISA; n = 27) and anti-GPI IgGs (Kd by bio-layer interferometry; n = 13) expressed from cloned genes that were identified by sequencing of TT-immunized mice and GPl-immunized mice, respectively, tested for binding to the immunogen. Boxes extend from the 25th to the 75th percentile, whiskers indicate the 10th to the 90th percentile and the line in the middle of the box is the median. h, Affinities of purified anti-TSPAN8 IgGs (Kd by flow cytometry; n = 3) expressed from cloned genes that were identified by sequencing of TSPAN8-immunized mice. See also Supplementary Table 3.

Article Snippet: Affinity was determined as follows: anti-TSPAN8 IgG antibodies that demonstrated specific binding to TSPAN8+ M300.19 cells were fluorescently labeled with Alexa Fluor 488 and incubated in serial dilutions with TSPAN8+ M300.19 cells in ice-cold FACS buffer (Ca2+, Mg2+-free PBS supplemented with 10% FBS) for 30 min on ice, and cell-bound fluorescence was quantified using a MACSQuant flow cytometer (Miltenyi Biotech).

Techniques: Sequencing, Expressing, Reverse Transcription, Emulsion, Amplification, Derivative Assay, Binding Assay, Clone Assay, Purification, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: bioRxiv

Article Title: An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis

doi: 10.1101/2020.12.29.424695

Figure Lengend Snippet: (A-C) Mammospheres from Snail-expressing HMLE cells were examined for morphology by brightfield microscopy or for ciliated cells by immunofluorescence for the indicated proteins by light sheet microscopy. Mammosphere-forming capacity was quantified for the indicated cells (n = 3 mean ± SEM). Representative results from 3 independent experiments are shown. Scale bars: 100 μm. (D) Organoid-forming capacity was determined for sorted MaSC-enriched basal cells in which GLIS2Cter was overexpressed (n = 3 mean ± SEM). Representative results from 3 independent experiments are shown. (E) Mammary glands from Glis2 +/+ or Glis2 -/- female mice (4 weeks old, n ≥ 5) were stained with Carmine Alum and ductal length was analyzed after whole mount preparation. Scale bar: 1 mm. (F) Paraffin sections from the mammary glands were stained with H&E or for the indicated protein. TSPAN8 signal intensity was measured per duct in all ducts of the sections analyzed. Representative images are shown. Scale bars: H&E 100 μm (inset: 2X), immunofluorescence 50 μm.

Article Snippet: The following primary antibodies were used: Arl13b (NeuroMab 73-287, 1:100), YFP (Cell Signaling 2956, 1:100), Slug (Cell Signaling 9585, 1:100), acetylated-tubulin (Cell Signaling 5335, 1:500), γ-tubulin (Sigma T5326, 1:50), SMA (Abcam ab21027, 1:100), E-cadherin (Cell Signaling 3195, 1:200), Cep170 (Fisher Scientific 10383523, 1:200), Krt8 (DSHB TROMA-I-s, 1 :1000), Krt14 (Biolegend 905301, 1:1000), Claudin 4 (Fisher Scientific 10303233, 1:200), Claudin 7 (Fisher Scientific 10537403, 1:200), Large T (Santa Cruz sc-20800, 1:200), VIM (Dako M0725, 1:200), TSPAN8 (Personal gift from Jane Visvader, 1:500).

Techniques: Expressing, Microscopy, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity

doi: 10.1126/sciadv.abh3375

Figure Lengend Snippet: ( A ) Schematic view of the experimental design. Stage 1 (PT, primary tumor) involved processing of tumors under physioxia and ambient air, flow cytometry characterization of cells, cell propagation, and reimplantation of cells into female FVB/N mice. Stage 2 (PL, primary line) involved flow cytometry characterization of cultured cells of stage 1. Stage 3 (TT, Transplanted Tumor) involved recharacterization of tumors obtained from cell implantation of stage 1. Stage 4 (TL, transplanted line) involved flow cytometry characterization of cells grown from stage 3 tumor. PT, Primary Tumor; PL, Primary Line; TT, Transplanted Tumor; TL, Transplanted Line. ( B ) Representative flow cytometry profile of tumor cells stained for antibodies against LGR5 and TSPAN8. Antibodies against lineage markers CD31-PE (phycoerythrin)/Cy7, CD45-PE/Cy7, and CD140a-PE/Cy7 were used to label endothelial cells, hematopoietic cells, and fibroblasts, respectively, and only lineage-negative cells were included in the analysis. ( C ) Quantitation of LGR5 + cells. Differences in LGR5 + cells between ambient air and physioxia are significant [ n = 3 to 6, one-way analysis of variance (ANOVA)]. ( D ) CD61/TSPAN8 staining patterns of tumor cells ( n = 3 to 6, one-way ANOVA). ( E ) Quantitation of TSPAN8 + cells. ( F ) Quantitation of CD61 + cells. ( G ) Tumor cells collected and processed at physioxia express higher levels of stemness-associated genes compared to ambient air ( n = 3, one-way ANOVA, P = 0.0004). * P < 0.05, ** P < 0.01, and *** P < 0.001 by ANOVA. ns, not significant.

Article Snippet: The antibodies used against mouse cells were CD31-PE/Cyanine7 (A14715) from Molecular Probes; CD45-PE/Cyanine7 (25-0451-82), EpCAM-APC (allophycocyanin) (17-5791-82), and CD29-FITC (fluorescein isothiocyanate) (11-0291-82) from Invitrogen; CD140a-PE/Cyanine7 (323508) from BioLegend; LGR5-PE (phycoerythrin) (FAB8240P), TSPAN8-APC (FAB6524A), and CD49f-PE (FAB13501P) from R&D Systems; and CD61-FITC (561911), CD274-PE (558091), CD24-APC (562349), and CXCR4-FITC (551967) from BD Pharmingen; isotype control antibodies used included PE/Cyanine7 (400522) and APC (400612) from BioLegend and PE (554689) and FITC (553971) from BD Pharmingen.

Techniques: Flow Cytometry, Cell Culture, Staining, Quantitation Assay

Journal: Science Advances

Article Title: Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity

doi: 10.1126/sciadv.abh3375

Figure Lengend Snippet: Note that tumor cells for both conditions were derived from the same tumor. ( A ) Phase-contrast images of PyMT tumor–derived cells with and without drug treatment ( n = 3, one-way ANOVA). ( B ) Quantitative measurement of cell survival data from (A). DMSO, dimethyl sulfoxide. ( C ) Cell proliferation rate at variable concentrations of drugs was measured using bromodeoxyuridine incorporation enzyme-linked immunosorbent assay ( n = 6, one-way ANOVA). Cancer cells used in this experimental series and in (A) were derived from a different PyMT + and Her2/Neu + mice. ( D ) Mice with PyMT tumor xenograft developed from tumor cells collected and processed under ambient air and physioxia were administered daily with lapatinib (Lap) (100 mg/kg of body weight) or vehicle control (VC) via oral gavage for 25 days ( n = 10, Student’s t test). Tumor growth was monitored every 5 days for 25 days. Treatment was initiated only after tumors reached similar size in both groups. ( E ) LGR5/TSPAN8 staining pattern of tumor cells from PyMT tumor xenografts from (D) ( n = 3, one-way ANOVA). APC, allophycocyanin. ( F ) Quantitation of LGR5 + cells. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 by ANOVA and Student’s t test.

Article Snippet: The antibodies used against mouse cells were CD31-PE/Cyanine7 (A14715) from Molecular Probes; CD45-PE/Cyanine7 (25-0451-82), EpCAM-APC (allophycocyanin) (17-5791-82), and CD29-FITC (fluorescein isothiocyanate) (11-0291-82) from Invitrogen; CD140a-PE/Cyanine7 (323508) from BioLegend; LGR5-PE (phycoerythrin) (FAB8240P), TSPAN8-APC (FAB6524A), and CD49f-PE (FAB13501P) from R&D Systems; and CD61-FITC (561911), CD274-PE (558091), CD24-APC (562349), and CXCR4-FITC (551967) from BD Pharmingen; isotype control antibodies used included PE/Cyanine7 (400522) and APC (400612) from BioLegend and PE (554689) and FITC (553971) from BD Pharmingen.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Control, Staining, Quantitation Assay