Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Overview of the workflow—B cells (or enriched ASCs) are isolated from mice (bone marrow or spleen) or human PBMCs. Cells are mixed with liquid BG-agarose at 37 °C and encapsulated into picolitre water-in-oil emulsion droplets using a flow-focusing junction. Droplets are collected on ice for agarose gelation and demulsified, creating stable hydrogel beads around each cell. The BG-agarose is converted into an antibody capture matrix by the addition of recombinant capture reagents that are fused to the SNAP-tag, an enzyme that reacts with BG moieties. During incubation, antibodies secreted by a single cell are captured in the hydrogel surrounding the cell. Cells that have secreted antigen-specific antibodies are identified with fluorescently labeled detection reagents (antigens, secondary antibodies and antibodies against cell-surface markers), sorted using flow cytometry and sequenced. Antibody sequences can be obtained within 4 days, and recombinant antibodies for testing are generated in 2 weeks. b , Agarose-based antibody capture matrix. Agarose is chemically modified to contain BG moieties that react covalently with the SNAP-tag. Single-domain antibodies (VHHs) against the constant region of antibody light chains are expressed as SNAP-tag fusions and immobilized in the BG-agarose hydrogel, creating the capture matrix. c , Antibody capture by BG-agarose hydrogel beads functionalized with VHH–SNAP. Antibody capture (anti-streptavidin mouse IgG) and antigen binding (streptavidin–GFP) were analyzed by flow cytometry. The plot shows at least 220 events per condition at a 5% contour level. d , Antibody secretion by single OVA-specific mouse bone marrow plasma cells. Representative confocal microscopy image from two independent experiments showing a single cell encapsulated in VHH-functionalized BG-agarose stained with fluorescently labeled OVA (AF555), anti-CD138 antibodies (AF647) and anti-mouse IgG antibodies (AF405). e , Sorting of OVA-specific mouse bone marrow plasma cells. Hydrogel beads containing plasma cells that secreted OVA-specific IgG were sorted by FACS (gated as live/FLAG + /CD138 + /IgM − /IgG + ). The plots show 10,168 (CD138 + ) and 411 (IgG + ) events at 2% contour level. f , Characteristics of mouse anti-OVA antibodies—variable domain genes (V and J), third complementarity-determining region amino acid sequences (CDR3s) and equilibrium dissociation constants ( K D ).

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Isolation, Emulsion, Recombinant, Incubation, Labeling, Flow Cytometry, Generated, Modification, Binding Assay, Clinical Proteomics, Confocal Microscopy, Staining

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Mouse immunization and analysis scheme. Bone marrow plasma cells (CD138 + ) were magnetically enriched and then used in our workflow. RBD-specific plasma cells were sorted with fluorescently labeled RBD-streptavidin tetramers. b , Sorting of RBD-specific mouse plasma cells. Cells were gated as live/CD138 + , and IgG-secreting RBD-specific plasma cells were sorted by FACS. The plots show 79,629 (CD138 + ) and 1,623 (IgG + ) events at 2% contour level. c , Overview of antibody sequences of sorted plasma cells. In total, 54 paired heavy- and light-chain sequences were obtained. The pie chart shows the 21 observed HV and LV gene combinations (HV–LV). HV–LV pairings are colored by the HV gene. Combinations that were characterized are shown in darker shades, while combinations that were not expressed are shown in lighter shades. The three most expanded expressed HV–LV combinations are highlighted with their CDRH3 amino acid sequence and frequency. d , Summary of anti-RBD ELISA. The plot shows the EC 50 with an antibody concentration range of 0.0002–400 nM. Antibodies that did not bind RBD at 400 nM are shown at an arbitrary EC 50 of 1,000 nM (gray diamonds). e , Characteristics of mouse anti-SARS-CoV-2 RBD antibodies with neutralizing capacity—variable domain sequences (V and J genes), third complementarity-determining region amino acid sequences (CDR3), equilibrium dissociation constants ( K D ) and IC 50 against WT SARS-CoV-2. f , In-tandem epitope binning experiment with mRBD1 and mRBD2. g , Crystal structure of mRBD2 with SARS-CoV-2 RBD (PDB: 8BE1 ). Top left, the RBD (green) in complex with mRBD2 Fab fragment (purple and pink for light and heavy chains) is superimposed with RBD complexed with ACE2 (gray; PDB: 6M0J ), showing how the Fab fragment overlaps significantly with ACE2. The main figure shows details of the RBD loop (green carbon atoms) binding to the CDRs of the mRBD2 Fab fragment.

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Clinical Proteomics, Labeling, Sequencing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: ( a ) Comparison of IgG signal originating from plasma cells in BG-agarose beads incubated with (+VHH) or without VHH–SNAP (−VHH; different experiment than shown in Fig. ). The plots show 2,536 (−VHH) and 14,606 (+VHH) events at 2% contour level. ( b ) Representative gating strategy for identifying RBD-specific plasma cells from mouse bone marrow (gated as live/CD138 + /IgM − /IgG + /RBD + ). ( c ) Anti-RBD ELISA. Workflow-derived antibodies are shown in black (binders) or blue (non-binders), a commercial positive control (clone 1035753, R&D Systems) is shown in red and an isotype control (mouse IgG1k, clone MG1-45; BioLegend) is shown in gray. The plot shows the mean of two technical replicates, representative of two independent experiments. ( d ) Neutralization of SARS-CoV-2 by mRBD1 and mRBD2. Wild-type SARS-CoV-2 (MOI = 0.01) was pre-incubated with a 3-fold dilution series of each antibody and then used to infect luminescent reporter cells. Levels of infection after 24 h were quantified as % of maximum luminescence. Mean values ± SD of three technical replicates are shown, representative of three independent experiments. IC 50 , half-maximal inhibitory concentration. ( e ) Determination of binding affinity of mRBD1 and mRBD2 by biolayer interferometry: mRBD1: K D = 0.864 ± 0.07 nM, χ = 0.0547, R = 0.991. mRBD2: K D = 4.78 ± 0.04 nM, χ = 0.1111, R = 0.9928.

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Comparison, Clinical Proteomics, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Positive Control, Control, Neutralization, Infection, Concentration Assay, Binding Assay

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: ( a ) Stimulation conditions and workflow for proof-of-concept in normal PBMCs. To obtain ASCs from normal PBMCs, B cells were stimulated with CD40L, IL-21 and anti-IgM antibodies. ( b ) IgG secretion of stimulated PBMCs encapsulated into hydrogel beads. The plot shows 66,173 events at a contour level of 2%; the gating strategy is shown in d . ( c ) Analysis of encapsulated stimulated PBMCs by epifluorescence microscopy. Hydrogel beads were stained with fluorescently labeled anti-CD38 (APC) and anti-human IgG antibodies (PE). The hydrogel bead boundary is shown as a dotted line. A representative image from one independent experiment is shown. ( d ) Gating strategy for identifying IgG-secreting human ASCs from stimulated B cells. ASCs were gated as DAPI − /CD14 − /CD3 − /IgD − /FLAG + /CD19 + /CD20 − /CD27 + /CD38 + (VHH–SNAP contains a FLAG tag).

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Epifluorescence Microscopy, Staining, Labeling, FLAG-tag

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Human anti-RBD antibody discovery. B cells were negatively selected from fresh or frozen PBMCs 7–9 days after the second BNT162b2 vaccine dose and then used in the workflow. RBD-specific ASCs were sorted with fluorescently labeled RBD-streptavidin tetramers. b , Sorting of RBD-specific human ASCs. Representative layout for sorting of IgG + /RBD + hydrogel beads obtained from the encapsulation of fresh B cells on d9 post vaccination. Hydrogel beads were gated as live/CD20 − /CD38 + /IgM − /IgG + . The plot shows 1,484 events at 2% contour level. c , Overview of sequences of human ASCs sorted with RBD. In total, 185 paired heavy- and light-chain sequences were obtained. The pie chart shows the observed HV–LV combination colored by the HV gene. HV–LV combinations that were expressed recombinantly are highlighted with their clone names and shown in a darker shade. d , Affinities ( K D ) of human anti-RBD antibodies. One antibody (gray diamond) did not show binding to RBD at the tested concentrations. e , Neutralization of WT and Omicron BA.1 SARS-CoV-2 by human anti-RBD antibodies. IC 50 was calculated from experiments performed in duplicate. Antibodies with no quantifiable neutralizing capacity at the highest concentration tested (100 μg ml −1 ) are shown at an arbitrary IC 50 of 1,000 μg ml −1 (gray diamonds). The ten antibodies with the lowest IC 50 against WT SARS-CoV-2 were also tested against Omicron BA.1 SARS-CoV-2. Antibodies neutralizing both variants are highlighted in red with their clone names. Neutralization of WT SARS-CoV-2 by the REGEN-COV (Ronapreve) mAb cocktail (casirivimab and imdevimab) was tested in the same assay (blue diamond). f , Sandwich epitope binning experiment with pairs of neutralizing human anti-RBD antibodies. In the network plot, the nodes show the antibody clones, the connections indicate pairwise blocking and the shaded areas indicate whether the antibodies belong to the same clonotype. Colors indicate the VH gene as in c .

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Labeling, Encapsulation, Binding Assay, Neutralization, Concentration Assay, Clone Assay, Blocking Assay

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Human anti-S1 antibody discovery. B cells were negatively selected from PBMCs 7 days after the second BNT162b2 vaccine dose and then used in the workflow. RBD/S1-specific ASCs were sorted with fluorescently labeled RBD- and S1-streptavidin tetramers. b , Index sorting of S1-specific human ASCs. Layout for sorting of IgG- or IgA-secreting ASCs isolated on day 7 post vaccination with fluorescently labeled S1- and RBD-streptavidin tetramers. Based on index sorting, the ratio of the S1/RBD fluorescence signal was calculated for all sequenced cells, and four sequences that corresponded to events with the highest S1/RBD fluorescence signal ratio were selected for expression (highlighted as red squares). The plot shows 719 events at a 5% contour level, and the percentage of events in each window is indicated. c , Characteristics of human anti-S1 antibodies—variable domain genes (V and J), third complementarity-determining region amino acid sequences (CDR3), equilibrium dissociation constants ( K D ) and IC 50 against WT SARS-CoV-2. En dashes (–) denote that binding or inhibition was not quantifiable at the tested concentrations. d , Anti-S1 and anti-RBD ELISA of anti-S1 antibodies. An RBD-binding positive control (human IgG1κ, clone AM001414 ; BioLegend) is shown in black, and an isotype control (human IgG1κ, clone QA16A12; BioLegend) is shown in gray. The table shows the EC 50 with an antibody concentration range of 0.0002–400 nM. The plots show mean values ± s.d. of two independent experiments performed in duplicate.

Article Snippet: We aim to increase sequencing output by optimizing single-cell sequencing protocols and by combining our sorting technology with the LIBRA-seq 10X Genomics workflow that uses DNA-barcoded antigens , which should also yield single-cell transcriptomic datasets.

Techniques: Labeling, Isolation, Fluorescence, Expressing, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Positive Control, Control, Concentration Assay

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Time assessments for digital cataract workflow and existing conventional workflow. *Time 1 was calculated as the sum of the mean times recorded for each step: (IOL Master) + (Pentacam) + (OCT) + (endothelial cell count). **Time 3: Digital transfer step not applicable in digital cataract workflow as it is automated. # Times 4 and 5: IOL axis marking is not applicable in the digital cataract workflow as it is done via the FORUM ® platform/CALLISTO eye.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques: Cell Counting

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Time Measurements at Various Steps in Site’s Existing Conventional and Digital Cataract Workflow

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques: Cell Counting

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Inter observer variability in the digital cataract workflow versus existing conventional workflow for preoperative assessments.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques:

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Time Savings Using a Digital Workflow versus a Conventional for Intraocular Lens Implantation in a Corporate Chain Hospital Setting

doi: 10.2147/OPTH.S439930

Figure Lengend Snippet: Overall time savings in digital cataract workflow versus existing conventional workflow.

Article Snippet: In conclusion, significant time savings at each step of cataract surgery planning were observed with the Zeiss digital cataract workflow compared with the existing conventional workflow.

Techniques:

Journal: bioRxiv

Article Title: A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

doi: 10.1101/2023.05.08.539575

Figure Lengend Snippet: Schematic overview of the cryo-/LP-CLEM workflow. Graphene liquid cell preparation is completely automated. The machine etches away the copper (7°C), transfers the graphene to a TEM grid via loop-assisted transfer and seals the GLCs by blotting away excess liquid. The reaction starts once the liquid pockets are sealed. Live fluorescence microscopy can be used to determine not only the location, but the specific time to image at high resolution, at which point the process is arrested by rapid vitrification. ROIs are located using cryo-fluorescence microscopy and hereafter they are imaged at nanometer resolution (cryo-TEM). The process is reinitiated inside the microscope by heating-up the grid using a cryo-holder, and the reaction dynamics are directly recorded (LP-TEM). To confirm that GLCs remain intact and retain liquid after TEM observation, fluorescence microscopy is used.

Article Snippet: After vitrification, the TEM grids were loaded into a universal TEM cryo-holder (349559-8100-010) using the ZEISS Correlative Cryo Workflow solution, which fit into the PrepDek® (PP3010Z, Quorum technologies, Laughton, UK).

Techniques: Fluorescence, Microscopy

Journal: bioRxiv

Article Title: A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

doi: 10.1101/2023.05.08.539575

Figure Lengend Snippet: Cryo-/LP-CLEM workflow to visualize crystallization processes inside a GLC. a) TEM overview imaged two days after thawing, overlaid with live-FM (green) to indicate the GLCs. Insert shows a high magnification LP-TEM image of the GLC in the blue box. b) SAED pattern taken at the position indicated by the black dashed circle in . Inner ring in the DP shows the contribution of the [100] plane on NaCl and outer ring the contribution of the graphene. c) Enlargement of the area marked by the orange box in were multiple GLCs are present (green, yellow and purple boxes) and a crystal that is not encapsulated by graphene (red box). D-f) Large crystal inside a GLC imaged at multiple time points after thawing (3d: 2 days; 3e: 5 days; 3f: 7 days) shows morphological changes. Graphene wrinkles (close yellow arrow), outline of the GLC (dotted yellow line) and the intensity gradient of the liquid surrounding the crystal (open yellow arrow) are visible at all time points. g) Two different but interconnected GLCs imaged two days after thawing (open and closed purple arrows). h) Condensation (solid arrow), and phase transformation (open purple arrow) observed within the pockets five days after thawing. I) Cubic NaCl crystal resulting from an Ostwald ripening process observed seven days after thawing j) Liquid pockets (green dashed circles) containing an amorphous-like phase. k) Nucleation within the amorphous phase contained in the GLCs (green striped circles). l) Dissolution of small crystals (top circle) and further crystallization (bottom circle) inside the GLCs. m) Crystal not encapsulated by graphene outlined by the red dotted line shows only minimum morphological changes five (3n) and seven (3o) days after thawing. Accumulative dose: a) 0.75e - /Å ; c, d, g, j, m) 0.15 e - /Å ; e, h, k, n) 0.30 e - /Å ; f, i, l, o) 0.45 e - /Å

Article Snippet: After vitrification, the TEM grids were loaded into a universal TEM cryo-holder (349559-8100-010) using the ZEISS Correlative Cryo Workflow solution, which fit into the PrepDek® (PP3010Z, Quorum technologies, Laughton, UK).

Techniques: Crystallization Assay, Transformation Assay