Journal: Biofabrication

Article Title: Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform

doi: 10.1088/1758-5090/ac32a5

Figure Lengend Snippet: Immunofluorescence (IF) of small airway epithelial population at the epithelial level (a) and at the epithelial-endothelial junction (c), (d). (a) DAPI (blue), α-tubulin (green), MUC5AC (yellow), CK5 (magenta). (b) xyz-view of DAPI (blue), α-tubulin (green), MUC5AC (yellow), and CK5 (magenta) co-stain to show pseudostratified columnar epithelium structure of the small airway layer. (c) maximum projection of epithelial layer only. α-tubulin (green), ACE2 (magenta) along the surface/higher z-stacks of small airway epithelium. (d) maximum projection at vascular layer. DAPI (blue), CD31 (yellow), ACE2 (magenta) along vasculature layer/lower z-stacks of small airway-capillary constructs. (e) live perfusion of small airway-capillary construct at timepoint t=0 min. The dye was introduced into the reservoirs of the left parent channel (input channel/vessel) and the white arrows indicate the entry point from the vascular network into the output channel/vessel. Taken with Opera Phenix. Scale bar = 100 μm unless otherwise indicated in images.

Article Snippet: For the 3D SAEC-capillary construct, AF700-conjugated MUC5AC (Novus Biologicals, NBP2–32732AF700), PE-conjugated cytokeratin 5/8 (Novus Biologicals, NBP2–47824PE), and AF488-conjugated α-tubulin (Cell Signaling Technology, 8058S) were used to identify small airway epithelial cells.

Techniques: Immunofluorescence, Staining, Construct

Journal: Biofabrication

Article Title: Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform

doi: 10.1088/1758-5090/ac32a5

Figure Lengend Snippet: Flow cytometric evaluation of cell types in 3D respiratory tissue construct and table with cluster frequencies. (a) bivariate plot of AQP5 and Sp-C in 3D alveoli-capillary construct. (b) bivariate plot of AQP5 and Sp-C on transwell platform, alveolar monoculture. (c) bivariate plot of AQP5 and Sp-C in submerged T-75 alveolar monoculture. (d) bivariate plot of a-tubulin and MUC5AC in 3D small airway-capillary construct. (e) bivariate plot of α-tubulin and MUC5AC on transwell platform, small airway monoculture. (f) bivariate plot of α-tubulin and MUC5AC in submerged, T-75 small airway culture. Grey = negative control; pink – stained sample. (g) bivariate plot of CD31 and vimentin in 3D respiratory construct. (h) bivariate plot of CD31 and vimentin of submerged T-75 monoculture for endothelial cells, fibroblasts and pericyte, contour plots of cell types overlaid. (i) Stacked column graph of cell populations from (a)-(f). VAMC = vascularized alveolar multi-chip; VBMC = vascularized bronchiolar multi-chip; AEC = alveolar epithelial cells; SAEC = small airway epithelial cells; TW = transwell. Blue = endothelial cells; red = fibroblasts; black = pericytes. FlowJo for compensation matrix calculation and gating analysis.

Article Snippet: For the 3D SAEC-capillary construct, AF700-conjugated MUC5AC (Novus Biologicals, NBP2–32732AF700), PE-conjugated cytokeratin 5/8 (Novus Biologicals, NBP2–47824PE), and AF488-conjugated α-tubulin (Cell Signaling Technology, 8058S) were used to identify small airway epithelial cells.

Techniques: Construct, Negative Control, Staining

Journal: Cancer Discovery

Article Title: Combinatorial BCL2 Family Expression in Acute Myeloid Leukemia Stem Cells Predicts Clinical Response to Azacitidine/Venetoclax

doi: 10.1158/2159-8290.CD-22-0939

Figure Lengend Snippet: LSC-like cells as defined by functional and transcriptomic parameters are predominantly located in the immature GPR56 + but not in the CD64 + CD11b + mature subpopulation. A, FACS gating strategy for mature, non-LSC, and LSC-like subpopulations. Displayed are AML bulk cells from primitive CD34 + ( NPM1 -wild-type), CD34 − ( NPM1 -mutated), and monocytic ( NPM1 -mutated) samples. B, Percentages of mature, non-LSC-like, and LSC-like subpopulations among bulk AML cells in 72 diagnostic AML samples sorted by the frequency of the mature population. C, Schematic overview of the experimental setup for xenotransplantation experiments and RNA sequencing (RNA-seq) of FACS-sorted subpopulations. D, Percentage of human leukemic engraftment obtained from mature, non-LSC, and LSC-like subpopulations of 14 AML samples at endpoints in the bone marrow of NSG mice. Each dot represents an individual mouse with the line marking mean engraftment levels. E, Mean percentage of human engraftment per mouse obtained from mature, non-LSC, and LSC-like subpopulations of 14 AML samples at endpoints in the bone marrow of NSG mice. Each dot represents an individual patient with AML with the line marking mean engraftment levels. Friedmann test was used to compare LSC-like with non–LSC-like and mature subpopulations. F, PCA plot of bulk RNA-seq data from LSC-like, non-LSC, and mature subpopulations from Prim-AML ( n = 14) and Mono-AML ( n = 9) annotated based on subpopulation and AML subclass. Each dot represents a population from one AML sample. G, LSC17 score in LSC-like, non-LSC, and mature subpopulations from Prim-AML ( n = 14) or Mono-AML ( n = 9) patient samples. LSC17 score was calculated for each AML sample as the mean expression of the 17 LSC signature genes. H–J, Normalized counts of BCL2 ( H ), MCL1 ( I ), and BCL2L1 ( J ) expression in LSC-like, non-LSC, and mature subpopulations from Prim-AML ( n = 14) or Mono-AML ( n = 9) patient samples. K, Schematic representation of the experimental setup used in L–O to measure intracellular BCL2, MCL1, and BCL-xL protein expression by flow cytometry. L and M, Mean fluorescence intensity (MFI) of BCL2 in ( L ) AML bulk and ( M ) LSC-like, non-LSC, and mature subpopulations from Prim-AML ( n = 11) or Mono-AML ( n = 7) patient samples. N and O, MFI of MCL1 in ( N ) AML bulk and ( O ) LSC-like, non-LSC, and mature subpopulations from Prim-AML ( N = 11) or Mono-AML ( N = 7) patient samples. P and Q, Representative tSNE plots of ( P ) AML26 (Prim-AML) and ( Q ) AML50 (Mono-AML) showing expression of CD64, CD34, GPR56, MCL1, BCL2, and BCL-xL. AML bulk is defined as mononuclear cells from patients with AML after the exclusion of dead cells, doublets, lymphocytes, and nucleated erythrocytes. Two-way ANOVA with Tukey correction for multiple comparisons test was used to compare groups of four, and Mann–Whitney test was used to compare groups of two unless specified otherwise. Each dot represents an AML patient sample with the line marking the mean unless specified otherwise. Fix, fixation; IC-staining, intracellular staining; Mut, mutated; NL, non-LSC; NSG, NOD. Prkdc scid . Il2rg null ; Perm, permeabilization; WT, wild-type. Parts of the figure were created with BioRender.com .

Article Snippet: Fixed and permeabilized cells were stained separately for anti-human-BCL2-PE (clone 124, Cell Signaling Technology, cat #26295S), anti-human-MCL1-PE (clone D2W9E, Cell Signaling Technology, cat #65617S), and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S), or together for anti-human-BCL2-AF647 (clone 124, Cell Signaling Technology, cat #82655), anti-human-MCL1-AF488 (clone D2W9E, Cell Signaling Technology, cat #58326) and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S).

Techniques: Functional Assay, Diagnostic Assay, RNA Sequencing Assay, Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY, Staining

Journal: Cancer Discovery

Article Title: Combinatorial BCL2 Family Expression in Acute Myeloid Leukemia Stem Cells Predicts Clinical Response to Azacitidine/Venetoclax

doi: 10.1158/2159-8290.CD-22-0939

Figure Lengend Snippet: Response to 5-AZA/VEN therapy in patients with AML can be predicted by MAC scoring in LSC-like cells. A, Schematic representation of the experimental design for B–G . Mononuclear cells of AML patient samples treated first-line with 5-AZA/VEN from three independently processed cohorts (cohort 1: n = 17, cohort 2: n = 18, and vohort 3: n = 24) were stained with surface antibodies, followed by intracellular staining of three BCL2 family proteins. MAC-Score was calculated based on normalized BCL2 family protein expression levels in LSC-like, non-LSC, mature, and total blast cells. B, Expression of BCL2, MCL1, and BCL-xL in LSC-like cells of patients with AML from cohorts 1 and 2 combined and associated 5-AZA/VEN therapy outcome. Protein expression is shown as MFI z-scores. C, MAC-Score in LSC-like cells of patients with AML from cohorts 1 and 2 combined and association to 5-AZA/VEN therapy outcome. D, Comparison of MAC-Score in LSC-like, non-LSC, mature, and total blast cells of patients with AML from cohorts 1 and 2 and association to 5-AZA/VEN therapy outcome. E, EFS of first-line 5-AZA/VEN AML patients from cohorts 1 and 2 combined with above and below median MAC-Score, BCL2 expression, MCL1 expression, or BCL-xL expression in LSC-like cells. F, MAC-Score in LSC-like cells of patients with AML from cohort 3 and associated 5-AZA/VEN therapy outcome. G, EFS of first-line 5-AZA/VEN AML patients from cohort 3 with above (>0.4) and below (<0.4) median MAC-Score in LSC-like cells. H, Schematic representation of the experimental design for I–J . Mononuclear cells of patients with relapsed/refractory AML who received 5-AZA/VEN as a salvage therapy (cohort 4: n = 23) were stained with surface antibodies, followed by intracellular staining of BCL2 family proteins. I, MAC-Score in LSC-like cells of patients with AML from cohort 4 and associated 5-AZA/VEN therapy outcome. J, EFS of salvage-treated 5-AZA/VEN AML patients from cohort 4 with above (>0.4) and below (<0.4) median MAC-Score determined in LSC-like cells. Each dot represents an AML patient sample with the line marking the mean unless specified otherwise. Mann–Whitney test was used to compare groups and log-rank test to compare therapy durations of AML patients. R/R, relapsed/refractory to standard induction. Parts of the figure were created with BioRender.com .

Article Snippet: Fixed and permeabilized cells were stained separately for anti-human-BCL2-PE (clone 124, Cell Signaling Technology, cat #26295S), anti-human-MCL1-PE (clone D2W9E, Cell Signaling Technology, cat #65617S), and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S), or together for anti-human-BCL2-AF647 (clone 124, Cell Signaling Technology, cat #82655), anti-human-MCL1-AF488 (clone D2W9E, Cell Signaling Technology, cat #58326) and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S).

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Cell Host & Microbe

Article Title: Memory profiles distinguish cross-reactive and virus-specific T cell immunity to mpox

doi: 10.1016/j.chom.2023.04.015

Figure Lengend Snippet: Phenotypical profile of VACV/MPXV tetramer + CD8 + T cells in HBDs and mpox patients (A) Representative flow plots showing VACV/MPXV tetramer staining on CD8 + T cells (left) and frequencies (right). (B) Frequencies of naive and memory T cell subsets within tetramer + CD8 + T cells in HBDs and convalescent donors. (C) UMAP visualization of tetramer + CD8 + T cells from HBDs and convalescent donors, and expression of individual markers colored by marker expression levels. (D) Frequency of HLA-DR + CD38 + , Ki-67 + , Granzyme B + CD8 + T cells, and CXCR3 + expression in HBDs vs. convalescent donors. (E) Representative flow plots of transcription factors expression within VACV/MPXV + CD8 + T cells in HBDs vs. convalescent donors (left) and quantification (right). (F) Frequency of T-bet + CX3CR1 + terminally differentiated (effector) cells in HBDs vs. convalescent donors. (G) Spearman correlation of marker expression with time after symptom onset. (H and I) Frequency of MPXV-specific T SCM cells identified with tetramers (H) or AIM assay (I) after mild vs. moderate mpox. (J) Frequency of TCF1 + cells and TCF1/T-bet ratio among tetramer + cells after mild vs. moderate mpox. In (A), (B), (D)–(F), and (H)–(J), Mann-Whitney test. In (G), Spearman rank correlation. See also Figure S4 .

Article Snippet: Anti-human TCF1 AF488 , CellSignaling , Cat# 6444S; RRID: AB_2199302.

Techniques: Staining, Expressing, Marker, MANN-WHITNEY

Journal: Cell Host & Microbe

Article Title: Memory profiles distinguish cross-reactive and virus-specific T cell immunity to mpox

doi: 10.1016/j.chom.2023.04.015

Figure Lengend Snippet:

Article Snippet: Anti-human TCF1 AF488 , CellSignaling , Cat# 6444S; RRID: AB_2199302.

Techniques: Recombinant, Staining, Software

Journal: Cell reports

Article Title: IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii

doi: 10.1016/j.celrep.2023.112147

Figure Lengend Snippet:

Article Snippet: IL-18 display levels were determined by staining with AF488-conjugated anti-Myc (Cell Signaling Technologies).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Infection, Plasmid Preparation, Software

Journal: Cell reports

Article Title: IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii

doi: 10.1016/j.celrep.2023.112147

Figure Lengend Snippet:

Article Snippet: AF488 anti-Myc , Cell Signaling Technology , D84C12 RRID:AB_2798045.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Infection, Plasmid Preparation, Software