αpd 1 antibody  (Bio X Cell)

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: Ultracentrifugation‐based exploratory pre‐screening reveals heterogeneous stromal and myeloid surface‐marker patterns across synovial fluid EV isolates from arthritis patients. (a) Schematic overview of synovial tissue and major cell populations present in active arthritis. (b) Workflow for ultracentrifugation (UC)‐based exploratory pre‐screening of synovial fluid (SF)‐EV marker profiles. (c) Western blot analysis of UC‐derived bulk SF‐EV preparations from patients with different forms of arthritis (total n = 13; RA n = 6, CPPD n = 4, Gout n = 1, OA n = 2) loaded by equal total protein amounts (50 µg per lane) and probed for CD90, PDPN, CD68, CD48, CD163, syntenin‐1 and human serum albumin. Cell lysates (CL; 2.5 µg per lane) from cultured synovial fibroblasts (FLS) and M1‐like macrophages (M1) were included as positive controls for antibody reactivity and to confirm cell‐type‐association. For each patient sample, the marker panels shown were derived from the same immunoblot. (d) Relative distribution of selected markers across donor‐derived SF‐EV preparations. Densitometric quantification of marker signals normalised to the corresponding syntenin‐1 signal, shown as the percentage contribution of normalised intensity across donor samples. CPPD, calcium pyrophosphate deposition (CPPD) arthritis; OA, osteoarthritis; RA, rheumatoid arthritis.

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques: Screening, Marker, Tissue, Western Blot, Analysis, Derivative Assay, Cell Culture

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: In Vitro validation of CD90‐ and PDPN‐directed immunocapture of synovial fibroblast‐derived EVs following SEC‐UF enrichment. (a) Schematic of the size exclusion chromatography‐ultrafiltration (SEC‐UF) workflow used to enrich EVs from conditioned medium (CM) of cultured synovial fibroblasts. (b) Representative transmission electron microscopy (TEM) images of SEC‐UF‐enriched EVs from two arthritis‐derived synovial fibroblasts (RA and OA donor). (c) Nano‐flow cytometry (nFCM) single‐vesicle surface staining of synovial fibroblast‐derived EVs for CD63, CD90 and PDPN under non‐fixed, non‐permeabilised conditions. Data are shown for two representative arthritis‐derived synovial fibroblast EV preparations (RA and OA). (d) Representative Western blot analysis of SEC‐UF‐enriched EVs and corresponding cell lysates (CL) from two arthritis‐derived synovial fibroblasts (RA and OA). Blots were probed for canonical EV markers (CD63, CD9, CD81, syntenin‐1, annexin‐1, flotillin‐1), fibroblast‐associated markers (CD90, PDPN) and the EV exclusion marker calnexin. (e) Representative Western blot analysis of cytosolic (C) and membrane‐enriched (M) protein fractions from cell and SEC‐UF‐enriched EV lysates of two arthritis‐derived synovial fibroblasts preparations (RA and OA). Blots were probed for CD90, PDPN and for canonical EV markers (CD63, CD9, syntenin‐1, annexin‐1, flotillin‐1). (f) Schematic of magnetic bead‐based immunocapture of SEC‐UF‐enriched EVs using nanosised beads coupled to anti‐CD90 or anti‐PDPN antibodies, or matched isotype control antibodies. Marker‐negative or non‐captured EVs were collected in the flowthrough (FT), followed by collection of a wash fraction (W). Bead‐bound marker‐positive EVs were recovered in the first eluate (E1), with a second eluate (E2) collected to assess residual carryover and elution efficiency. (g) Representative TEM images of E and FT fractions following CD90‐ or PDPN‐directed immunocapture; arrows indicate EVs. (h) Representative Western blot analysis of EV fractions obtained after CD90‐ and PDPN‐directed immunocapture, including the EV input control (EV CTRL), fractions E1‐E2, W, FT and corresponding isotype‐bead control. Blots were probed for CD90, PDPN and canonical EV markers (CD63, CD9, syntenin‐1, annexin‐1, flotillin‐1). (i) Densitometric quantification of immunocapture outcomes from Western blot analyses of n = 3 independent experiments using SEC‐UF‐enriched EVs from synovial fibroblasts of different arthritis patients, shown as log 2 relative density ratios for proteins of interest after normalisation to syntenin‐1. Paired E‐versus‐FT comparisons were assessed by paired Student's t ‐test (ns, not significant; * p < 0.05; ** p < 0.005; *** p < 0.001). Lines connect matched E‐FT pairs. P1 and P2 denote two PDPN signal populations resolved by immunoblot.

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques: In Vitro, Biomarker Discovery, Derivative Assay, Size-exclusion Chromatography, Cell Culture, Transmission Assay, Electron Microscopy, Flow Cytometry, Staining, Western Blot, Analysis, Marker, Membrane, Control

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: CD90‐directed immunocapture selectively retrieves CD90+ EVs from mixed EV preparations and synovial fluid‐spiked samples. (a) Schematic of immunocapture from a defined mixed EV preparation generated by combining SEC‐UF‐enriched EVs from synovial fibroblasts and THP‐1 macrophages at a 1:1 ratio. CD90 + EVs are captured and eluted (E1, E2), while unbound EVs are collected as flowthrough (FT) and wash (W). (b) Representative Western blot analysis of E and FT fractions from the CD90‐directed immunocapture using the mixed EV preparation, probed for CD90, PDPN, CD68 and for canonical EV markers CD63, CD9 and syntenin‐1. An isotype‐bead control was included to assess capture specificity. EV input controls (bulk SEC‐UF‐enriched EVs prior to immunocapture) from each source are shown. Densitometric quantification of CD90‐directed immunocapture outcomes from Western blot analyses of n = 3 independent experiments using SEC‐UF‐enriched EVs from synovial fibroblasts of different arthritis patients, shown as log 2 relative density ratios for CD90, PDPN and CD68 after normalisation to syntenin‐1. Paired E‐versus‐FT comparisons were assessed by paired Student's t ‐test (** p < 0.005; *** p < 0.001). Lines connect matched E‐FT pairs. (c) Schematic of CD90‐directed immunocapture following spiking of synovial fibroblast‐derived SEC‐UF‐enriched EVs into a pre‐treated synovial fluid sample lacking detectable endogenous CD90 signal. The fractions E1, E2, W and FT were collected; unbound EVs in FT were subsequently purified and enriched by SEC‐UF as indicated. (d) Representative Western blot analysis of E1 and E2, FT and W fractions from synovial‐fluid spiked‐in experiments, probed for CD90, PDPN and for canonical EV markers CD63, CD9 and syntenin‐1, with an isotype‐bead control and EV input control (spiked SEC‐UF EV population). Densitometric quantification of immunocapture outcomes shown as log 2 relative density ratios for CD90 signal intensity normalised to syntenin‐1. No statistical assessment was performed from the paired E1 versus FT, as no CD90 signal was detectable in the FT fraction (nd, not detected).

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques: Generated, Western Blot, Analysis, Control, Derivative Assay, Purification

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: SEC‐UF enrichment enables CD90‐ and PDPN‐directed immunocapture of stromal‐associated EV populations from arthritic synovial fluid. (a) Schematic of the synovial fluid pre‐treatment and SEC‐UF EV enrichment workflow, followed by magnetic bead‐based immunocapture using nanosised antibody‐coupled beads. (b) Representative transmission electron microscopy (TEM) images of two independent synovial fluid preparations with and without SEC‐UF processing, illustrating the reduction of visually apparent background material and recovery of EVs following the enrichment workflow. (c) Western blot analysis of SEC‐UF‐enriched SF‐EV preparations from three arthritis patients (Patients 5, 7 and 8), probed for stromal‐associated markers (CD90, PDPN), selected myeloid/immune‐associated markers (CD68, CD48, CD163) and syntenin‐1. Cell lysates (CL; 2.5 µg per lane) from cultured synovial fibroblasts and M1‐like macrophages were included as positive controls for antibody reactivity and to confirm cell‐type‐association. For each patient sample, all marker panels shown were derived from the same immunoblot. (d) Western blot analysis and densitometric quantification of EV fractions obtained after immunocapture in SEC‐UF‐enriched SF‐EVs using anti‐CD90 or anti‐PDPN beads, shown for patients 5, 7 and 8. Eluate (E), flowthrough (FT) and wash (W) fractions are displayed on the same immunoblot; total recovered protein material from each fraction was analysed. Stromal‐associated signals (CD90, PDPN; red frames) and selected myeloid/immune‐associated signals (CD163, CD68, CD48; blue frames) are indicated, together with probed canonical EV markers (CD63, CD9, syntenin‐1) and human serum albumin. For quantification, band intensities were normalised to syntenin‐1 and expressed as log 2 relative density ratios. Paired E‐versus‐FT comparisons for CD90 and the two PDPN isoforms (P1 and P2) were assessed by paired Student's t ‐test (ns, not significant; * p < 0.05; ** p < 0.005; *** p < 0.001). Lines connect matched E–FT pairs. No statistical assessment was performed for the selected myeloid/immune‐associated markers because CD68, CD48 and CD163 signals were not detectable in the corresponding E fractions (nd, not detected).

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Analysis, Cell Culture, Marker, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: Surfaceome‐informed cell‐of‐origin profiling links eluate‐ and flowthrough‐enriched SF‐EV proteins to stromal‐ versus immune‐associated cell signatures. (a) Volcano plot of differential protein abundance between the CD90‐immunocaptured synovial fluid (SF) EV eluate fraction (E) and the non‐captured CD90 − flowthrough fraction (FT). Protein's meeting significance and effect‐size thresholds (FDR < 0.05; |log 2 FC| ≥ 1.5) are highlighted (red dots); selected membrane‐associated proteins are annotated as indicated. (b) Box plots showing log 2 ‐normalised abundance values for representative proteins enriched in the E fraction (upper panel) or FT fraction (lower panel), as indicated. Box plots show median and interquartile range (IQR); whiskers indicate the 5th–95th percentiles. (c) Distribution of significantly enriched surface proteins across curated cell‐type signature (CTS) groups. Proteins were restricted to those with cell surface protein consensus (SPC) scores 4–3–2 and assigned to 10 CTS groups; counts are shown for E‐ versus FT‐enriched proteins, as indicated. (d) Venn diagrams comparing E‐ and FT‐enriched protein sets (SPC score 4–3–2) with published synovial fibroblast and synovial tissue single‐cell transcriptomic reference datasets (Korsunsky et al. ; Edalat et al. ), as cited in the main text). (e, f) Projection of E‐ and FT‐enriched proteins onto two independent single‐cell gene expression atlases: (e) Broad Institute Single Cell Portal (SCP; AMP Phase I RA synovial tissue dataset; Zhang et al., ) and (f) Tabula Sapiens. Dot plots/UMAP representations show the distribution of mapped proteins (as shown in Panel d) across annotated cell types as displayed in each atlas. In Panel e, dot size denotes the proportion of cells within a given cell type annotation expressing the corresponding gene, and dot colour density indicates the average expression level, as displayed. In Panel f, coloured circles in UMAP plots depict annotated cell type clusters. (g) Schematic summary of membrane proteins enriched in the CD90‐immunocaptured SF‐EV subpopulation (Eluate fraction) versus membrane proteins enriched in the heterogenous pool of non‐captured CD90 − SF‐EV populations (flowthrough fraction). Figure created with BioRender.com.

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques: Profiling, Quantitative Proteomics, Membrane, Tissue, Single Cell, Gene Expression, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Enrichment and Proteomic Profiling of Stromal‐Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size‐Exclusion Chromatography Coupled to CD90‐Directed Immunocapture

doi: 10.1002/jev2.70353

Figure Lengend Snippet: Selected CD markers ( n = 150) detected in synovial fluid EVs from arthritis patients and grouped by cell‐type association.

Article Snippet: The membrane was blocked in blocking buffer (5% milk in TBS with 0.05% Tween20 [TBS‐T]) for 1 h at RT, followed by three washing steps for 5 min in washing buffer (TBS‐T) and an overnight incubation with primary antibodies at 4°C (CD90 (CST‐13801S‐Rabbit; 1:1000), PDPN (CST‐9047‐Rabbit; 1:1000), CD68 (UltraMAB‐UM870047‐Mouse; 1:2000), CD63 (Invitrogen‐10628D‐Mouse; 1:1000), CD9 (Invitrogen‐10626D‐Mouse; 1:1000), CD81 (Invitrogen‐10630D‐Mouse; 1:1000), CD163 (CST‐93498‐Rabbit; 1:1000), Syntenin‐1 (Abcam‐ab133267‐Rabbit; 1:1000), Flotillin‐1 (BD‐610820‐Mouse; 1:1000), Annexin‐1 (BD‐610067‐Mouse; 1:5000), Calnexin (CST‐2679S‐Rabbit; 1:1000), CD48 (CST‐29499T‐Rabbit; 1:1000)).

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Influence of LCN2 on the growth of lung cancer in mice. (A) Simplified flowchart outlining the establishment of an anti-PD-L1 immunotherapy tolerance model in mice with lung cancer. (B) Western blot analysis of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice. (C) Immunohistochemical detection of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice (scale bar, 100 µm). (D) Establishment of a model for anti-PD-L1 immunotherapy in LCN2-silenced mice. (E) Evaluation of LCN2 mRNA expression in tumor tissues after LCN2 gene intervention in mice. (F) Assessment of LCN2 protein expression in tumor tissues following LCN2 gene intervention in mice. (G) Tumor size in LCN2 gene intervention mice (n=10 in each group). *P<0.05, **P<0.01 and ***P<0.001. Unpaired two-tailed Student's t-test was used for (B) and (C) and one-way ANOVA with Tukey's multiple comparisons test was used for (E) and (G). LCN2, lipocalin 2; PD-L1, programmed death-ligand 1; sh-, short hairpin; NC, negative control; COL1A1, collagen α-1(I) chain; THY1, Thy-1 membrane glycoprotein; i.p., intraperitoneal.

Article Snippet: The following primary antibodies were used: Rabbit anti-LCN2 (cat. no. ab125075; 1:100; Abcam), rabbit anti-COL1A1 (cat. no ab34710; 1:15; Abcam), rabbit anti-Thy-1 membrane glycoprotein (THY1) (cat. no 13801; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-E-cadherin (cat. no 3195; 1:400; Cell Signaling Technology, Inc.), rabbit anti-Vimentin (cat. no. 5741; 1:4,000; Cell Signaling Technology, Inc.), rabbit anti-IL-6 (cat. no. ab6672; 1:400; Abcam), rabbit anti-TNF-α (cat. no. ab307164; 1:1,000; Abcam), rabbit anti-Ki-67 (cat. no. ab16667; 1:200; Abcam) and rabbit anti-cleaved caspase-3 A (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Two Tailed Test, Negative Control, Membrane