cd31 microbead kit  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Oxidative Stress Susceptibility, Complement Dysregulation, and Metabolic Reprogramming in CFH Y402H Patient-Derived Choriocapillaris Endothelial Cells

doi: 10.64898/2026.06.18.733111

Figure Lengend Snippet: A ) Schematic diagram of the stepwise differentiation of iPSCs into CECs. B ) Bright-field images: day 0; iPSCs, day 5; embryoid bodies, day 8; differentiating cells. The CD31-positive endothelial cells were isolated (day 14) and expanded from the heterogeneous population using the CD31 MicroBead Kit. Representative brightfield images of CD31-negative and CD31-positive cells at day 20. Scale bars = 100 μm. C ) Quantitative RT-PCR showing enrichment of CEC markers in the CD31 + enriched cell populations in both HR and ISO cultures. Data are presented as mean ± SEM, n=3, unpaired t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: On day 14, after cell treatment with TrypLe Express (Life Technologies, 12604013), CD31-positive cells were isolated from a single cell suspension with the CD31 MicroBead Kit (Miltenyi Biotec, 130-091-935) according to the manufacturer’s protocol.

Techniques: Isolation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Oxidative Stress Susceptibility, Complement Dysregulation, and Metabolic Reprogramming in CFH Y402H Patient-Derived Choriocapillaris Endothelial Cells

doi: 10.64898/2026.06.18.733111

Figure Lengend Snippet: CECs were stained for choriocapillaris endothelial cell markers: CD31, RGCC, CA4, PLVAP (red). Nuclei were counterstained with Hoechst (blue). Positive staining was assessed by comparing to a secondary antibody-only control, scale bars = 50 μm.

Article Snippet: On day 14, after cell treatment with TrypLe Express (Life Technologies, 12604013), CD31-positive cells were isolated from a single cell suspension with the CD31 MicroBead Kit (Miltenyi Biotec, 130-091-935) according to the manufacturer’s protocol.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Schematic of directed differentiation protocol to generate hiEndos. (B) Phase contrast images during the indicated day of differentiation. White dashed square represents region magnified in insets. Scale bar: 50um. (C-E) RT-qPCR for the indicated primers using RNA isolated from cells on each day of differentiation, purified hiEndos at P0 and P5, and HUVECs. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. (F) Flow cytometry of hiEndos using antibodies against the human endothelial lineage markers CD31 and CD144 (right) and isotype controls (left), N=4 biological replicates from distinct differentiations. (G) Immunostaining of hiEndos for human CD31 (green, left) and CD144 (red, right). Hoechst stains nuclei (blue). Scale bar 10um. N=3 biological replicates. (H) Capillary tube formation assay on 3D Matrigel. Scale bar 50um. N=4 biological replicates. (I-J) Human CD31 and acetylated LDL uptake in hiEndos measured by flow cytomtetry (I) or fluorescence microscopy (J). Scale bar 50um. N=8 biological replicates. (K) Schematic of experiment to test the impact of Notch (DAPT) and TGF-beta (SB431542) inhibition on hiEndo expansion (top). Quantification of CD31 and CD144 double-positive cells by flow cytometry (left) and cumulative hiEndo yield (right) at each passage grown in the indicated media conditions. N=2 experimental replicates from independent wells of the same differentiation, representative data shown from 3 independent differentiations. (L) Quantification of total hiEndo yield per input hiEndo in each media. ** p<0.01 using one-way Anova and Tukey’s multiple comparison test. (M) RT-qPCR of arterial and venous markers after serial passaging in SD medium. N=4 experimental replicates from independent wells of the same differentiation, representative results from two independent differentiations. ** p<0.01, *** p<0.001, **** p<0.0001, one-way Anova and Tukey’s multiple comparison test. Abbreviations: hiPSC (human induced pluripotent stem cell), CHIR (CHIR99021), P (passage), HUVEC (human umbilical vein endothelial cells), AcLDL (acetylated low-density lipoprotein), SB (SB431542).

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Quantitative RT-PCR, Isolation, Purification, Flow Cytometry, Immunostaining, Capillary Tube Formation Assay, Fluorescence, Microscopy, Inhibition, Comparison, Passaging

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A-C) Fold change in the expression of indicated transcripts in hiPSC-derived cells (BU3 clone) on each day of hiEndo differentiation, in purified hiEndos at P0 and P5, and in HUVECs (RT-qPCR). N=4 experimental replicates from independent wells of the same differentiation. (D) Quantitation of hiEndo differentiation efficiency on day 5 of differentiation across 12 distinct differentiations. (E) Flow cytometry of human CD31, human CD144, and isotype controls pre- and post-MACs purification of hiEndos on day 5 of the differentiation. N=1. (F) Phase contrast microscopy of BU3 hiEndos showing cobblestone morphology. Scale bar 50um. (G) Flow cytometry of human CD31, human CD44, and isotype controls in BU3 hiEndos, the plot is representative of 4 experimental replicates of independent wells from the same differentiation. (H) Quantification of the percent of CD31/CD144 double positive hiEndos by flow cytometry at each passage. N=1 differentiation (BU1). (I) Phase contrast microscopy of BU1 hiEndos at sequential passages. Scale bar 50um. Results are representative of 2 experimental replicates of independent wells from the same differentiation. (J) RT-qPCR analysis of fold change in transcript expression of markers of fibroblast and smooth muscle identity in hiEndos at sequential passages. N=2 experimental replicates of independent wells from the same differentiation. **** p<0.0001 by one-way Anova with Tukey’s multiple comparisons.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Expressing, Derivative Assay, Purification, Quantitative RT-PCR, Quantitation Assay, Flow Cytometry, Microscopy

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Schematic of the general strategy for the competitive lung reconstitution assay. (B) Cartoon depiction of experiment to test feasibility of competitive lung reconstitution assay by transplanting defined ratios of EGFP+ and dsRED+ cells that were all pre-treated with BMP9+SB431542 (top). Flow cytometry gated on human CD31+ post-transplant hiEndos 10-days following injection and analyzed for dsRED and EGFP expression (bottom). N=1 animal per condition. (C) Quantitation of the contribution of SB- vs BMP9+SB-treated hiEndos in the mouse lung 2 weeks following transplantation as determined by flow cytometry across 2 distinct experiments. N=4 animals per experiment, p-value determined by Student’s paired two-tailed t-test. (D) Left lung lobe immunofluorescence microscopy to identify EGFP+ and dsRED+ hiEndos 2-weeks following transplantation of BMP9 pretreated hiEndos. hiEndos are largely present in the alveolar space (top inset) as opposed to larger vessels (bottom inset). White dotted lines indicate region of inset. A airway, V vessel. N=3 animals. (E-G) Immunofluorescence confocal microscopy of recipient mouse lung tissue sections 2 weeks following transplantation of mixtures of EGFP+ and dsRED+ hiEndos. 3D reconstruction of Z-stacks shows each indicated antibody-stained fluorochrome together with immunostaining for human CD31 (hCD31, E, white), mouse Cd31 (mCd31, F, white), or podoplanin (Pdpn, G, red). Insets (G) show close apposition of Pdpn and EGFP+ hiEndo. Hoechst stains nuclei (blue). Scale bars (E-G) 10um. N=4 animals.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Reconstitution Assay, Flow Cytometry, Injection, Expressing, Quantitation Assay, Transplantation Assay, Two Tailed Test, Immunofluorescence, Microscopy, Confocal Microscopy, Staining, Immunostaining

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Flow cytometric analysis of mouse lung single cell digests from animals 5-weeks post-transplantation with EGFP-labeled, BMP9+SB-treated hiEndos and stained with either an isotype or human CD31 BV421-conjugated antibodies. (B) UMAP representation of post-transplant BU1 hiEndos showing expression of key endothelial lineage markers. (C and D) As in (B) except showing expression of TMEM100 (C) or VEGFA (D) expression in post-transplant hiEndos.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Single Cell, Transplantation Assay, Labeling, Staining, Expressing

Journal: bioRxiv

Article Title: A chimeric human-mouse lung vascular model using induced pluripotent stem cells reveals insights into the pathogenesis of BMPR2 -related pulmonary hypertension

doi: 10.64898/2026.04.29.721664

Figure Lengend Snippet: (A) Schematic describing the generation of two distinct hiPSC lines from two individuals with pulmonary hypertension and the heterozygous pathogenic BMPR2 p.(C118W) variant and syngeneic gene-corrected controls. (B) Flow cytometry for hCD31 and hCD144 on day 5 of differentiation shows increased hiEndo yield in BMPR2 C118W/+ cells that is quantified in (C; N=4 distinct differentiations per line). (D) Growth assay shows increased cumulative cell yield in BMPR2 C118W/+ hiEndos compared to gene-corrected controls N=2 experimental replicates of independent wells from the same differentiation, representative data shown from three distinct differentiations. **** p<0.0001 determined by 2-way Anova with Sidak’s multiple comparison test. (E) Immunostaining of hiEndos treated with vehicle or BMP9 for 4 hours and stained with antibodies raised against phospho-SMAD1/5/9 (PSMAD, red). Scale bar 40um. Quantification of nuclear PSMAD fluorescent intensity normalized to total-SMAD1/5/9 intensity is shown to the right. **** p<0.0001 by one-way Anova and Tukey’s multiple comparison test. N=3 experimental replicates from independent wells of the same differentiation, representative data shown from three distinct differentiations. (F) RT-qPCR analysis of ID2 expression in BMPR2 C118W/+ and +/+ hiEndos treated with BMP9 for 24 hours. N=4 experimental replicates from independent wells of the same differentiation, representative data shown from three distinct differentiations. **** p<0.0001 by one-way Anova and Tukey’s multiple comparison test. (F) Schematic for competitive transplantation between BMPR2 C118W/+ and gene-corrected hiEndos. (G) Quantification of the relative contribution of each genotype to the total transplanted hiEndo population 2- and 3-weeks post-transplantation. * p<0.05, **** p<0.0001, Student’s paired two-tailed t-test. Each dot represents an individual animal. (H) Whole-mount fluorescent image of lungs following competitive transplant of BMPR2 C118W/+ (dsRED) and +/+ (EGFP) hiEndos. Scale bar 2mm. (I) 3D reconstruction of confocal Z stacks of transplanted hiEndos from the competitive lung reconstitution assay showing BMPR2 C118W/+ (dsRED) and +/+ (EGFP) hiEndos stained with antibodies raised against human CD31 (hCD31, white, top) and mouse CD31 (mCD31, white, bottom). Arrows in insets show red blood cells contained in lumen formed by hiEndos. Experiment replicated with N=3 animals. Scale bar 10um.

Article Snippet: The cell pellet was then incubated with anti-human CD31 (1:10, Miltenyi, #130-091-935) and anti-human CD144 microbeads (1:10, Miltenyi, #130-097-857) diluted in EGM2 medium (50uL total volume per well of a 6-well plate, Lonza, #CC-3162) for 20 minutes.

Techniques: Variant Assay, Flow Cytometry, Growth Assay, Comparison, Immunostaining, Staining, Quantitative RT-PCR, Expressing, Transplantation Assay, Two Tailed Test, Reconstitution Assay