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Figure 3. Identifying the disease driver population in CAVD (A) Representative image of FACS-sorted aortic VICs cultured in normal media (NM), high phosphate media (PM), or osteogenic media (OM). Unsorted VICs (mix), sorted CD44high, and sorted CD44+ cells calciﬁed under OM and PM conditions (PM > OM), but not in NM; Alizarin Red staining at day 21 (n = 3). (B) Multicolor ﬂow cytometry shows co-localization of CD44high VICs with CD29+, <t>CD59+,</t> and CD73+ staining, with lower staining of CD45; n = 3 donors. (C) Immunohistochemistry of areas proximal to calciﬁcation show more abundant staining of markers CD44, CD29, CD59, and CD73 than in areas more distal to the calciﬁcation (n = 3). (D) Sorted VICs population CD44highCD29+CD59+CD73+CD45low cells readily differentiated into calcifying osteoblast-like cells in OM (Alizarin Red staining) and fat droplet-containing adipocyte-like cells in AM (Oil Red O) similar to MSCs differentiation in the same media. Alcian blue staining of chondrocyte-like DDP VICs in CM. Mixed (MIX) unsorted aortic VICs show no comparable differentiation in OM, CM, or AM; n = 4 donors. (E) Normal aortic VICs, CD44highCD29+CD59+CD73+CD45low sorted (SORT) and unsorted (MIX) mixed, differentiates on OM, CM, and AM with no considerable change at 2 weeks. Adult normal human dermal ﬁbroblasts are used as normal ﬁbroblast reference.

Cd59 Fluidigm 3173009b, supplied by fluidigm, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/cd59+fluidigm+3173009b/Anti-Human+CD59+(p282+(H19))-173Yb/pm35417712-478-68-69
Average 94 stars, based on 1 article reviews

cd59 fluidigm 3173009b - by Bioz Stars, 2026-10

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1) Product Images from "A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling."

Article Title: A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.

Journal: Cell reports

doi: 10.1016/j.celrep.2022.110685

Figure Legend Snippet: Figure 3. Identifying the disease driver population in CAVD (A) Representative image of FACS-sorted aortic VICs cultured in normal media (NM), high phosphate media (PM), or osteogenic media (OM). Unsorted VICs (mix), sorted CD44high, and sorted CD44+ cells calciﬁed under OM and PM conditions (PM > OM), but not in NM; Alizarin Red staining at day 21 (n = 3). (B) Multicolor ﬂow cytometry shows co-localization of CD44high VICs with CD29+, CD59+, and CD73+ staining, with lower staining of CD45; n = 3 donors. (C) Immunohistochemistry of areas proximal to calciﬁcation show more abundant staining of markers CD44, CD29, CD59, and CD73 than in areas more distal to the calciﬁcation (n = 3). (D) Sorted VICs population CD44highCD29+CD59+CD73+CD45low cells readily differentiated into calcifying osteoblast-like cells in OM (Alizarin Red staining) and fat droplet-containing adipocyte-like cells in AM (Oil Red O) similar to MSCs differentiation in the same media. Alcian blue staining of chondrocyte-like DDP VICs in CM. Mixed (MIX) unsorted aortic VICs show no comparable differentiation in OM, CM, or AM; n = 4 donors. (E) Normal aortic VICs, CD44highCD29+CD59+CD73+CD45low sorted (SORT) and unsorted (MIX) mixed, differentiates on OM, CM, and AM with no considerable change at 2 weeks. Adult normal human dermal ﬁbroblasts are used as normal ﬁbroblast reference.

Techniques Used: Cell Culture, Staining, Cytometry, Immunohistochemistry

Figure 4. Single-cell RNA sequencing (scRNA-seq) of in vitro 2-week calciﬁcation assay with MSC-NM, MSC-OM, MIX-NM, MIX-OM, DDP- NM, and DDP-OM cells (A) Single-cell InDrops scRNA-seq of in vitro calciﬁcation assay. MSCs and VICs 2-week culture in either OM or NM. Cells were detached and immediately sorted for single-cell capture, cDNA library synthesis, and sequencing. (B) tSNE plot showing k-means clusters of MSCs. (C) tSNE plot with MSC-NM and MSC-OM grouping. (D) tSNE plot showing k-means clustering of VICs: VIC-NM and DDP-OM separation. (E) tSNE plot showing k-means clusters of VICs and how MIX-NM, MIX-OM, DDP-NM, and DDP-OM separate. (F) MSC k-clusters expression heatmap of increased DGEs from fold change R2.0 of k4 versus k2 cluster at FDR; q % 0.05. (G) VICs k-clusters expression heatmap of increased DEGs from fold change R1.5 of k4 versus (k1 + k2) clusters at FDR; q % 0.05. DDP, sorted CD44highCD29+CD59+CD73+CD45low VICs; DEGs, differentially expressed genes; FDR, false discovery rate; MIX, mixed unsorted VICs; MSCs, mesenchymal stem cells; NM, normal media; OM, osteogenic media; QC, quality control; tSNE, t-distributed stochastic neighbors embedding; VICs, valvular interstitial cells, .

Figure Legend Snippet: Figure 4. Single-cell RNA sequencing (scRNA-seq) of in vitro 2-week calciﬁcation assay with MSC-NM, MSC-OM, MIX-NM, MIX-OM, DDP- NM, and DDP-OM cells (A) Single-cell InDrops scRNA-seq of in vitro calciﬁcation assay. MSCs and VICs 2-week culture in either OM or NM. Cells were detached and immediately sorted for single-cell capture, cDNA library synthesis, and sequencing. (B) tSNE plot showing k-means clusters of MSCs. (C) tSNE plot with MSC-NM and MSC-OM grouping. (D) tSNE plot showing k-means clustering of VICs: VIC-NM and DDP-OM separation. (E) tSNE plot showing k-means clusters of VICs and how MIX-NM, MIX-OM, DDP-NM, and DDP-OM separate. (F) MSC k-clusters expression heatmap of increased DGEs from fold change R2.0 of k4 versus k2 cluster at FDR; q % 0.05. (G) VICs k-clusters expression heatmap of increased DEGs from fold change R1.5 of k4 versus (k1 + k2) clusters at FDR; q % 0.05. DDP, sorted CD44highCD29+CD59+CD73+CD45low VICs; DEGs, differentially expressed genes; FDR, false discovery rate; MIX, mixed unsorted VICs; MSCs, mesenchymal stem cells; NM, normal media; OM, osteogenic media; QC, quality control; tSNE, t-distributed stochastic neighbors embedding; VICs, valvular interstitial cells, .

Techniques Used: RNA Sequencing, In Vitro, cDNA Library Assay, Sequencing, Expressing, Control

Virus:

Article Title: A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER 159Tb-Anti-human NANOG HMS Lederer Core facility N/A; RRID: not found 162Dy-Anti-human ki-67 Fluidigm 3162012B; RRID: not found 163Dy-Anti-human TGFb Fluidigm 3163010B; RRID: AB_2888928 166Er-Anti-human CD44 Fluidigm 3166001B; RRID: AB_2744692 168Er-Anti-human CD73 Fluidigm 3168015B; RRID: AB_2810249 169Tm-Anti-human aSMA HMS Lederer Core facility N/A; RRID: not found 170Er-Anti-human CD105 HMS Lederer Core facility N/A; RRID: not found 172Yb-Anti-human pS6 Fluidigm 3172008A; RRID: not found 173Yb-Anti-human CD59 Fluidigm 3173009B; RRID: not found 174Lu-Anti-human OCN HMS Lederer Core facility N/A; RRID: not found 176Yb-Anti-human Sortilin HMS Lederer Core facility N/A; RRID: not found Donkey anti-goat IgG AF594 Thermo Fisher A-11058; RRID: AB_2534105 Bacterial and virus strains No bacterial or virus strains used N/A N/A; RRID: not found Biological samples Human CAVD post-operative aortic valve leaflets BWH N/A Human Normal aortic valve leaflets, post-autopsy BWH N/A HAoVIC, Normal valve leaflets cells from human donor # 1 Lonza CBS-58004A; RRID: not found hVIC, Normal valve leaflets cells from human donor # 2 Lonza 00225974; RRID: not found Chemicals, peptides, and recombinant proteins Formalin Thermo Fisher Scientific SF96-20 Picrosirius red solution VWR KT037 Picric acid N/A SHUR/MountTM Liquid Mounting Medium, TBS VWR 15148-062 VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories H-1200; RRID: AB_2336790 Harris Modified Method Hematoxylin Stains Thermo Fisher Scientific SH30-4D; SH30-500D Eosin Y 1% VWR 10143-130 Radioimmunoprecipitation assay buffer (RIPA) buffer Cell Signaling Technologies 9806 Protease inhibitor cocktail Sigma Aldrich P8340-5ML Chloroform(Approx. .. 0.75% Ethanol as Preservative/Molecular Biology), Thermo Fisher Scientific BP1145-1 Methanol, OptimaTM LC/MS Grade, Fisher Chemical Thermo Fisher Scientific A456-1 Urea Sigma Aldrich U4884-500G Thiourea ReagentPlus , R99.0% Sigma Aldrich T7875-500G UltraPureTM 1M Tris-HCI, pH 8.0 Thermo Fisher Scientific 15568025 Triethylammonium bicarbonate Sigma Aldrich 17902-100ML RapiGest SF Waters Technologies Corporation 186001861 Tris(2-carboxyethyl)-phosphine hydrochloride Sigma -Aldrich 77720 Iodoacetamide Thermo Fisher Scientific A39271 Lysyl Endopeptidase Fujifilm Wako 125-05061 Fisher chemical Water, OptimaTM LC/MSGrade Thermo Fisher Scientific W6-1 Trifluoroacetic acid for HPLC, R99.0% Sigma-Aldrich 302031-10X1ML OASIS HLB extraction cartridge Waters Technologies Corporation 186000383 Acetonitrile Thermo Fisher Scientific A955-1 (Continued on next page) Cell Reports 39, 110685, April 12, 2022 e2

Recombinant:

Modification:

Radio Immunoprecipitation:

Protease Inhibitor:

other:

Article Title: A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.
Article Snippet: 0.75% Ethanol as Preservative/Molecular Biology), Thermo Fisher Scientific BP1145-1 Methanol, OptimaTM LC/MS Grade, Fisher Chemical Thermo Fisher Scientific A456-1 Urea Sigma Aldrich U4884-500G Thiourea ReagentPlus , R99.0% Sigma Aldrich T7875-500G UltraPureTM 1M Tris-HCI, pH 8.0 Thermo Fisher Scientific 15568025 Triethylammonium bicarbonate Sigma Aldrich 17902-100ML RapiGest SF Waters Technologies Corporation 186001861 Tris(2-carboxyethyl)-phosphine hydrochloride Sigma -Aldrich 77720 Iodoacetamide Thermo Fisher Scientific A39271 Lysyl Endopeptidase Fujifilm Wako 125-05061 Fisher chemical Water, OptimaTM LC/MSGrade Thermo Fisher Scientific W6-1 Trifluoroacetic acid for HPLC, R99.0% Sigma-Aldrich 302031-10X1ML OASIS HLB extraction cartridge Waters Technologies Corporation 186000383 Acetonitrile Thermo Fisher Scientific A955-1 (Continued on next page) Cell Reports 39, 110685, April 12, 2022 e2

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