Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: HPCs generated in iSTC-Hp-spheroids can undergo maturation to erythroid cells, macrophages, or T lymphocytes using established cell maturation protocols (A) Schematic outline illustrating the use of Mart1-iPSC cells for iSTC-Hp-spheroid formation and subsequent erythroid cell, macrophage, and T lymphocyte differentiation. (B) Frequencies of CD34 + CD43 + and CD34 + CD45 + cells in Mart1-iPSC-derived iSTC-Hp-spheroids on day 13. Values represent mean ± SD from 3 independent experiments. (C–E) Mart1-iPSC-derived HPCs cultured in the erythroid differentiation condition for 15 days were harvested to characterize erythroid cell properties. The color of cell pellets changed to red (C), and typical erythroid lineage markers, such as CD71 and GPA, could be detected by flow cytometry analysis (D). Globin expression patterns were determined by relative RNA expression of α- and ζ-globin (α-globin series) and ε-, γ-, and β-globin (β-globin series) (E). Values represent mean ± SD from 3 independent experiments. (F–I) Macrophage differentiation from Mart1-iPSC-derived CD34 + cells. (F) Macrophage surface markers were analyzed by flow cytometry (gray; isotype control). (G) Giemsa stain of macrophage cytospin. Scale bar: 50 μm. (H) Phagocytosis assay of fluorescein isothiocyanate (FITC)-labeled zymosan A particles (gray; isotype control). (I) DHR assay of reactive oxygen species production by Mart1-iPSC-derived macrophages in response to stimulation with PMA (bottom panel); unstimulated macrophage control is also shown (top panel). (J–L) T lymphocytes generated from Mart1-iPSC-derived HPCs. (J) After 22 days of T cell differentiation, CD4 + CD8 + double-positive T cells could be detected by flow cytometry analysis, most of which were CD3 + Mart1-tetramer + cells. (K) Mart1-iPSC-derived CD4 + CD8 + double-positive T cells could be induced to CD8αβ single-positive T cells by stimulation with Mart1 peptide-primed T2 cells. (L) Mart1-iPSC-derived CD8 + single-positive T cells released interferon γ (INFγ) and tumor necrosis factor α (TNF-α) in response to Mart1 peptide-pulsed T2 cells. T cell responses without T2+Mart1 peptide (no target) or without Mart1 peptide (T2+DMSO) are also shown. Values represent mean ± SD from 3 independent experiments, ∗∗∗∗p < 0.0001.

Article Snippet: CD4 Micro-Bead Kit, human , Miltenyi Biotec , Catalog No: 130-045-101.

Techniques: Generated, Derivative Assay, Cell Culture, Flow Cytometry, Expressing, RNA Expression, Control, Giemsa Stain, Phagocytosis Assay, Labeling, Cell Differentiation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: The iSTC-Hp-spheroid system can be performed in a xeno-free condition (A) Schematic outline of screening a xeno-free condition for the iSTC-Hp-spheroid system. (B) Representative flow cytometry analysis of CD34, CD43, and CD45 on GFP + cells in iSTC-Hp-spheroids cultured in the xeno-free medium for 13 days. GFP + cells are gated for analysis. (C) Comparison of the frequency of CD34 + CD43 + cells and CD34 + CD45 + cells in iSTC-Hp-spheroids between culturing in FBS-based medium vs. xeno-free medium. GFP + cells are gated for analysis. Values represent mean ± SD from 3 independent experiments; ns, not significant. (D) Representative flow cytometry analysis of T lineage cell markers, including CD5, CD7, CD4, and CD8, on cells differentiated from HPCs generated in xeno-free condition. Cells were harvested on day 22 of T cell differentiation. GFP + cells are gated for analysis. (E) Quantifications of flow cytometry analysis in (D). Values represent mean ± SD from 3 independent experiments. (F) Representative flow cytometry analysis of CD3 + cells in CD4 + CD8 + cells during the T cell differentiation. GFP + cells are gated for analysis. (G) Quantifications of flow cytometry analysis in (F). Values represent mean ± SD from 3 independent experiments. (H) Frequencies of CD3 + cells in CD4 + CD8 + cells on day 32 of T cell differentiation. GFP + cells are gated for analysis. Horizontal bars represent mean value from 3 independent experiments; ns, not significant. (I) TCR sequence analysis showing the diversity of rearranged TCR genes in NCRM5-EGFP-derived CD4 + CD8 + CD3 + T cells isolated from day 32 of the T cell differentiation.

Article Snippet: CD4 Micro-Bead Kit, human , Miltenyi Biotec , Catalog No: 130-045-101.

Techniques: Flow Cytometry, Cell Culture, Comparison, Generated, Cell Differentiation, Sequencing, Derivative Assay, Isolation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet:

Article Snippet: CD4 Micro-Bead Kit, human , Miltenyi Biotec , Catalog No: 130-045-101.

Techniques: Recombinant, Giemsa Stain, Modification, Cell Differentiation, Software

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: GATA-1-high BM progenitors show strong priming for erythroid lineage (A) BM aspirate is ficolled and enriched for CD34 + cells before gating for CD34 + /CD38 + cells and selecting GATA-1-high population (top ∼8%) and remaining GATA-1-expressing cells (denoted as GATA-1 mid/low) (bottom ∼87%). (B) InTAC-seq genome coverage plots at GATA1 and SPI1 loci for GATA-1-high and -mid/low BM progenitors. (C) Heatmap of chromVAR deviation scores across GATA-1 high and mid/low BM progenitors for top 50 most variable motifs. (D) MA plot of log2 fold change in accessibility between GATA-1 high and mid/low BM progenitors versus log2 mean number of reads in consensus peaks. Peaks with significant changes in accessibility are highlighted in red or blue. (E) Most significantly enriched TF motifs in differentially accessible peaks between GATA-1-high and -mid/low BM progenitors calculated using Fisher’s test. (F) (Top) Average accessibility of GATA-1 motif sites across all consensus ATAC-seq peaks binned by GATA-1 motif score. Accessibility is defined here as the area under the curve of a plot of bias-corrected, normalized Tn5 insertions centered at GATA-1 motif sites, integrated from −50 to +50 bp and excluding the TF footprint from −10 to +10 bp. (Bottom) Difference in GATA-1 motif accessibility between GATA-1 high and mid/low samples normalized to the accessibility in the GATA-1 mid/low population for each motif score bin. (G) UMAP of previously published and annotated BM scATAC dataset with Seurat clusters manually annotated as key BM populations. (H) Normalized GATA1 gene expression across BM progenitors in UMAP space (expression derived from scRNA-seq data integrated with scATAC-seq data). (I) Bulk BM progenitor InTAC-seq data simulated as scATAC counts and projected onto scATAC UMAP space.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Expressing, Gene Expression, Derivative Assay

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: High-dimensional, single-cell proteomic analysis of BM progenitors identifies TF and surface-marker trends in erythroid commitment (A) BM aspirate is ficolled and enriched for CD34 + cells before staining with antibodies to surface marker panel and key TF to capture single-cell protein abundance using mass cytometry. High-dimensional data with over 1 million cells were used to delineate heterogeneity in BM progenitors and find surface-marker surrogates for GATA-1 TF abundance for further functional validation. (B) Force-directed layout (ForceAtlas2) of density downsampled (to 250,000 cells) CD45 + -gated BM progenitors colored by manually gated populations. (C) Normalized marker expression of key surface and TF proteins (in orange) across force-directed layout. Orange arrow denotes GATA-1-high region in single-cell map. (D) Leiden clusters of BM progenitors (resolution = 1) visualized on force-directed layout. (E) Barplot of frequency of manually gated BM progenitor populations across 11 Leiden clusters. (F) Normalized diffusion pseudotime calculation visualized on force-directed layout with trajectory from HSCs to erythroid-primed progenitors across Leiden clusters 3, 1, and 11 (in red). (G) Row-normalized heatmap of median marker abundance at 100 bins across diffusion pseudotime-aligned trajectory. Orange font and arrows indicate TF protein trends, and bold font with black arrows indicate key surface marker trends in trajectory. (H) Column-normalized heatmap of mutual information scores calculated on cells in Leiden clusters 2, 7, and 8 and normalized across key TF and surface-marker pairs.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Marker, Staining, Quantitative Proteomics, Mass Cytometry, Functional Assay, Biomarker Discovery, Expressing, Diffusion-based Assay

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: Surface-marker-defined BM population surrogate for high GATA-1 protein abundance clonally enriches for erythroid lineage (A) Spearman correlation plot of surface markers and GATA-1 abundance in BM progenitors as measured by mass cytometry. (B) Top 8% of GATA-1-expressing cells in CD34 + /CD38 + BM progenitors gated as GATA-1-high BM cells, and remaining GATA-1-mid/low cells used for subsequent analysis. (C) Boxplots of normalized surface-marker abundance of GATA-1-high BM progenitors (top ∼8% of expression) and GATA-1-mid/low BM progenitors (bottom ∼87% of expression) from mass cytometry. (D) Violin plots of GATA-1 protein abundance in manually gated target populations (as defined by CD71 + , CD84 + , CD33 – ), CD123 – MEP populations, and in other BM progenitor populations. (E) Boxplot of clonal differentiation frequency of target population and CD123 - MEP population to different lineages/population types across 4 biological replicates. (p values calculated using Student's t test)

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Marker, Quantitative Proteomics, Mass Cytometry, Expressing

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: High GATA-1 protein abundance delineates epigenetic program for erythroid commitment in RBC developmental trajectory (A) BM aspirate is ficolled and enriched for CD34 + cells before gating for CD123 – MEP population (CD34 + /CD38 + /CD10 – /CD45RA – /CD123 – ) and selecting high- (25%–40%) and mid- (∼lower 30%) GATA-1-expressing cells within each compartment. (B) GATA-1-high cells from CD34 + /CD38 + and CD123 – MEP compartments InTAC-seq data were simulated as scATAC counts and projected on scATAC UMAP space. (C) Putative erythropoiesis trajectory constructed from HSCs to late erythoid populations and overlaid on scATAC UMAP. (D) Heatmap of top variable TFs by ChromVAR deviation scores across constructed erythroid trajectory with the projected position of GATA-1-high InTAC-seq samples indicated in red as the point of GATA-1-high overlap, in blue as the before point, and in green as the after point. Top: line plot of InTAC-seq-denoted GATA-1-high-simulated scATAC cells as binned across pseudotime. (E) Top 20 genes significantly enriched (of fold change 2 and above) in integrated scRNA-seq data between the 3 bins, before, at, and after GATA-1-high overlap points in trajectory. (F) Summary schematic of continuous differentiation to erythrocytes in BM with downregulation of lymphoid/myeloid TF activity and gene expression programs and upregulation of erythroid TF activity and gene expression programs. High GATA-1 protein abundance overlaps epigenetic program shift to erythroid lineage commitment in human BM.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Quantitative Proteomics, Expressing, Construct, Activity Assay, Gene Expression

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet:

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Mass Cytometry, Software

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Lentiviral splicing reporter and RNA sequencing-based detection of splicing alterations in pAML (A) Schematic of lentiviral dual-fluorescence MAPT splicing reporter. (B) Representative image of Kasumi-1, a pAML cell line, stably transduced with the pCDH-EF1-IRES-Puro MAPT splicing reporter lentiviral vector. Left: confocal fluorescence microscopic image of lentiviral splicing reporter GFP expression (scale bar, 50 μm). Right: confocal fluorescence microscopic image of lentiviral splicing reporter RFP expression (scale bar, 50 μm). (C) FACS analysis of GFP and RFP mean fluorescence intensity (MFI) in the live cell population of CD34 + selected pAML patient samples (n = 4 biological replicates). (D) FACS analysis of GFP MFI in the live cell population of CD34 + selected pAML samples versus CD34 + cord blood (CB) samples (Student’s t test, p = 0.029; n = 4 biological replicates). (E) Whole-transcriptome (RNA-seq)-based quantification of CD44 splice isoforms in pAML (n = 9, blue) versus pNL (n = 3; red) HSCs (Student's t test, p < 0.05). (F) RNA-seq-based quantification of CD47 splice isoform expression in pAML (blue) versus pNL (red) HSCs (Student's t test, CD47-201 p = 0.3766; CD47-202 p = 0.0019; CD47-204 p = 0.0167; CD47-205 p = 0.0134). See also Figure S4 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: RNA Sequencing, Fluorescence, Stable Transfection, Transduction, Plasmid Preparation, Expressing

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Single-cell proteogenomic sequencing analysis of clonal heterogeneity in pAML Single-cell proteogenomic profiling was performed using the MissionBio Tapestri system to identify clonal somatic mutations and proteomic alterations. Uniform manifold approximation and projection (UMAP) plots (left) are shown from three separate pAML patients and were generated using eight antibody-oligo conjugates (CD3, CD7, CD11b, CD34, CD38, CD45, CD56, and ROR1) and a DNA mutation panel covering 45 myeloid genes with 312 amplicons. (A) pAML 266; correspondence of immunophenotyping and single nucleotide variant (SNV) analysis with ROR1, CD7, CD38, and CD34 cell surface protein expression is shown in a heatmap with each row associated with a single cell (right). (B) pAML 451; correspondence of immunophenotyping and SNV analysis with CD34, CD38, CD7, and ROR1 cell surface protein expression is shown in a heatmap with each row associated with a single cell (right). (C) pAML 678; correspondence of immunophenotyping and SNV analysis with CD7, CD38, CD34, and ROR1 cell surface protein expression is depicted (right). See also Figure S2 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Sequencing, Generated, Mutagenesis, Variant Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: RBFOX2 downregulation and reversion to an embryonic stem cell splicing program in pAML (A) RBFOX2 protein expression levels were quantified in pAML (n = 5 biological replicates) and cord-blood-derived HSCs (n = 3 biological replicates) and hematopoietic progenitor cells (HPCs; pAML, n = 5; pNL, n = 4 biological replicates) by FACS analysis as shown by MFI (Student’s t test HSC [left] p = 0.04; HPC [right] p = 0.06). (B) RNA-seq-based cytoscape network analysis of RBFOX2 and embryonic stem cell (ESC) specific alternative splicing program-related transcripts. Nodes are colored according to log fold change. (C) Volcano plot highlighting RBFOX2 target genes that show differential exon usage in pAML (false discovery rate, FDR < 0.05; percent spliced in, PSI > 0.1). A negative PSI reflects exon inclusion in pAML, while a positive PSI reflects ES in pAML versus pNL cells. (D) Heatmap of exon expression levels after unsupervised clustering of RBFOX2 target genes in pAML HSCs and HPCs (n = 9 and n = 8, respectively; blue) compared with pNL (n = 3 and n = 6, respectively; red). (E) Cytoscape network analysis of differentially spliced RBFOX2 target genes implicated in “Pathways in Cancer” in pAML HPCs. The top 200 ES events in this comparison were used as seeds for network propagation on the STRING high-confidence interactome. Differential expression log fold change is mapped to node color. Triangles depict seed list genes, and circles represent associated genes. See also Figure S5 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Expressing, Derivative Assay, RNA Sequencing, Alternative Splicing, Comparison, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Rebecsinib inhibition of pAML LSC survival and self-renewal (A) Colony assay of CD34 + cord blood cells in long-term stromal co-cultures following lentiviral shRNA control (shControl, n = 7 biological replicates) or shRNA knockdown of RBFOX2 (shRBFOX2, n = 7 biological replicates; Student’s t test, p = 0.03). Data represent mean ± SD. (B) Percent colony survival and self-renewal (replating) of CD34 + cord blood samples after treatment with 100 or 1,000 nM of Rebecsinib normalized to vehicle-treated controls. Mean colony percentages ± SD are shown for each treatment condition (n = 7 biological replicates). Statistical analysis was performed by one-way ANOVA (survival [left] 100 nM Rebecsinib shControl versus shRBFOX2 p = 0.003, 1,000 nM Rebecsinib shControl versus shRBFOX2 p < 0.001; self-renewal [right] 100 nM Rebecsinib shControl versus shRBFOX2 p < 0.001, 1,000 nM Rebecsinib shControl versus shRBFOX2 p < 0.001). (C) Survival and self-renewal of CD34 + cells derived from pAML (n = 6 biological replicates) or cord blood (CB) (n = 7 biological replicates) in long-term stromal co-cultures. Data represent mean ± SD for each treatment condition. Statistical analysis was performed by one-way ANOVA (survival [left] 0.1 μM Rebecsinib pAML versus CB p = 0.001, 1 μM Rebecsinib pAML versus CB p = 0.001, 10 μM Rebecsinib pAML versus CB p = 0.001; self-renewal [right] 0.1 μM Rebecsinib pAML versus CB p = 0.001, 1 μM Rebecsinib pAML versus CB p = 0.001, 10 μM Rebecsinib pAML versus CB p = 0.001). (D) Self-renewal of CD34 + pAML (n = 6 biological replicates) versus adult dnAML (n = 9 biological replicates) treated with Rebecsinib. Data represent mean ± SD for each treatment condition. Statistical analysis was performed by one-way ANOVA (pAML versus adult dnAML p < 0.001). (E) Representative fluorescence microscopic imaging analysis of the pAML cell line Kasumi-1 cells stably transduced with the lentiviral pCDH-EF1-IRES-Puro MAPT dual-fluorescence splicing reporter treated with (left) DMSO (scale bar, 200 μm) and (middle) 100 nM Rebecsinib (scale bar, 200 μm). Right: the ratio of RFP/GFP was quantified using Volocity software (Quorum Technologies) following Rebecsinib treatment and normalized to DMSO vehicle controls (n = 1). (F) FACS analysis of RFP/GFP MFI in the live cell population of CD34 + selected pAML patient samples treated with 100 nM Rebecsinib for 72 h (n = 6 biological replicates, Student's t test, p = 0.016). (G) FACS analysis of RFP/GFP MFI in MOLM13 (a leukemia cell line derived from a 20-year-old male) cells stably transduced with the lentiviral pCDH-EF1a-IRES-Puro MAPT dual-fluorescence splicing reporter and treated in vitro with Rebecsinib (100 nM) versus DMSO (n = 3 technical replicates, data represent mean ± SEM, Student's t test, p < 0.0001). (H) MOLM13-MAPT splicing reporter stably transduced cells were engrafted into irradiated NSG-SGM mice, treated with vehicle (n = 3 biological replicates) or Rebecsinib (10 or 20 mg/kg; n = 2, n = 3 technical replicates), and sacrificed 24 h post-treatment. The ratio of RFP/GFP MFI in live, human CD45 + cells in bone marrow, spleen, and peripheral blood was quantified by FACS analysis using a MACS Quant instrument and FlowJo software. Data represent mean ± SEM for each treatment condition. Statistical analyses were performed using unpaired, two-tailed Student’s t test (bone marrow [left] vehicle versus 20 mg/kg p = 0.0013, peripheral blood [middle] vehicle versus 20 mg/kg p = 0.0488, spleen [right] vehicle versus 20 mg/kg p < 0.0001). See also Figure S6 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Inhibition, Colony Assay, shRNA, Control, Knockdown, Derivative Assay, Fluorescence, Imaging, Stable Transfection, Transduction, Software, In Vitro, Irradiation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet:

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Virus, Clinical Proteomics, Recombinant, Staining, Generated, Software, RNA Binding Assay

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

Figure Lengend Snippet: SARS-CoV-2 spike-specific mAb binding profiles (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors and the subset of those that expressed S-binding BCRs. Right: cells from three donors expressing S-binding BCRs and sorted to recover principally those that did not bind RBD. (B) Summary of all productive mAbs (recombinant human IgG1) screened by ELISA (with recombinant S ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown next to each chart. To the right of the charts for the two sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding mAbs for those subjects from whom at least ten paired-chain BCR sequences were recovered. (C) Binding to a panel of S proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA and by association with S expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for S + sorted (left) and S + RBD − sorted cells (right). Left panel, 157 clones bound to S and an additional one bound to only RBD but not S. Pink indicates ELISA screens. Blue indicates cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heatmaps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of VH and VL mutation counts in mAbs that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; nonparametric Kruskal-Wallis multiple comparison. Horizontal lines show mean ± SEM. See also Figure S1 .

Article Snippet: B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 μg/ ml flag-tagged S protein or mixture of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD ( ) on ice for 30 min.

Techniques: Binding Assay, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Clone Assay, Labeling, Mutagenesis, Comparison

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

Figure Lengend Snippet: Sorting strategy for SARS-CoV-2-specific memory B cells and characterization of monoclonal antibodies, related to Figure 1 (A) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 S-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD − IgM-IgG + CD27 + and finally on S (B) Representative flow cytometry plots showing S-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the S gate. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK293T cells. Cells were gated on DAPI − GFP + population. (D) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (E) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Figure 1 C for the screening color scheme. (F) Bar graph of Log 10 (EC 50 ) of antibodies targeting RBD, NTD and S2 using ELISA and cell-based assay. EC 50 (μg/mL), RBD (n = 23), NTD clusters (n = 15) and S2 (n = 15). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Paired nonparametric Wilcoxon test. Data are mean values ± SEM (G) Dot plot of Log 10 (EC 50 ) of antibodies in the indicated bins by cell-based assay. Antibodies are from subjects G32 and C41, sorted with S. Each dot represents one monoclonal antibody. EC 50 (μg/mL), 13-39 days (n = 13), 40-63 days (n = 8). No significance; nonparametric Mann-Whitney test. Data are mean values ± SEM.

Article Snippet: B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 μg/ ml flag-tagged S protein or mixture of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD ( ) on ice for 30 min.

Techniques: Bioprocessing, Flow Cytometry, Binding Assay, Control, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Cell Based Assay, MANN-WHITNEY

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

Figure Lengend Snippet:

Article Snippet: B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 μg/ ml flag-tagged S protein or mixture of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD ( ) on ice for 30 min.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Saline, Random Hexamer, Reverse Transcription, Transfection, Luciferase, Plasmid Preparation, Flow Cytometry, Software