anti human cd45 alexa fluor 647 direct conjugate  (Bio-Rad)

Journal: Nature Communications

Article Title: ICAM-1/CD18-mediated sequestration of parasitized phagocytes in cortical capillaries promotes neuronal colonization by Toxoplasma gondii

doi: 10.1038/s41467-025-58655-z

Figure Lengend Snippet: A Experimental set up. Freshly egressed GFP-expressing T. gondii ( Tg ) tachyzoites (PRU-GFP, 2 × 10 5 cfu) were inoculated ip in mice and organs were collected 3 or 6 days post-inoculation (dpi). B Representative flow cytometry plots of GFP vs forward scatter (FSC) show percentage of tachyzoite-associated PBMCs (GFP + ) in unchallenged and infected mice 3 and 6 dpi ( n = 3 mice). C Representative micrographs of brain cortex at 3 or 6 dpi. Mice were injected iv with α-CD45-Alexa 647 prior to organ extraction. Arrows indicate leukocyte (CD45 + , red)-associated tachyzoites (GFP + , green). Dotted lines delimit cortical capillaries. Asterisk indicates tachyzoite (GFP + ) non-associated to a leukocyte (CD45 - ). Lower panel insets show magnification of the leukocyte- associated tachyzoites. Scale bars: 10 µm. D Cartoon shows microsurgical procedure, as detailed in Methods, with injection of cell suspensions (DC /Tg ) into the internal carotid artery (ICA), followed by brain extraction and tissue sectioning (50 µm thickness). Common carotid artery (CCA) and external carotid artery (ECA) are indicated. Created in BioRender. Pairoto, M. (2025) https://BioRender.com/e26i498 . E Experimental set up. CFSE or CMTMR pre-labeled DCs were challenged in vitro with T. gondii (RH-GFP, MOI 1; ME49-RFP, MOI 2; CFSE-prelabelled CTG, MOI 2) to obtain a DC infection frequency of ∼50%. Infected DCs (~20 × 10 6 DCs / ~ 10 × 10 6 cfu Tg ) were slowly (5 min) inoculated into the brain circulation via the ICA as detailed in Methods. Brains were extracted 16 h post-inoculation (hpi). F Confocal micrographs, with corresponding 3D surface analyses, show the localization of Tg type I RH (GFP, green)-infected DCs (CMTMR, red), Tg type II ME49 (RFP, red)-infected DCs (CFSE, green) and Tg type III CTG (CFSE, green)-infected DCs (CMTMR, red) in relation to the vascular marker Evans blue (cyan). Arrows indicate infected DCs magnified in the insets, respectively. Scale bars: 10 µm. G 3D surface analysis as in ( F ) of microvessel (cyan) with infected DC (red) with T. gondii vacuoles (green). Microvessel lumen diameter (5,04 µm) and vascular branching point are indicated. H , I Luminal diameter ( H ) and distance to nearest vascular branching point ( I ) of cortical microvessels containing T. gondii type I, II, and III-infected DCs, respectively. In box plots, center line indicates median. Box limits: 25 th and 75 th percentiles. Whiskers: maximum and minimum values. Data are from 86 ( Tg I), 61 ( Tg II), 47 ( Tg III) -infected DCs per condition from three independent experiments ( Tg I n = 4 mice; Tg II, Tg III n = 3 mice). J . Bar graph shows mean ( ± SEM) numbers of T. gondii type I, II or III-infected DCs related to cortical area, from 30 ( Tg I), 28 ( Tg II) and 27 ( Tg III) cortical sections per condition from 3 independent experiments ( n = 3 mice per condition). K Bar graph shows the relative attachment frequency of T. gondii type I- or type II-infected DCs to polarized brain endothelial cells (bEnd.3) in vitro. Data are expressed as mean ( ± SEM) from three independent experiments ( n = 3 biological replicates). Statistical analyses: ( H – J ) Kruskal–Wallis test followed Dunn’s post-hoc test ( K ) 2-tailed unpaired Student’s t -test, numeric p -values are indicated, ns: non-significant, p > 0,05. Source data are provided as a Source Data file.

Article Snippet: When indicated, anti-mo CD45 Alexa fluor 647-conjugated antibody (R&D Systems, Cat# FAB3507R, Clone 319211, Lot# 1726103) was injected iv (0,4 mg/kg) 10 min before euthanasia.

Techniques: Expressing, Flow Cytometry, Infection, Injection, Extraction, Labeling, In Vitro, Marker

Journal: Experimental and Therapeutic Medicine

Article Title: Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer

doi: 10.3892/etm.2024.12583

Figure Lengend Snippet: Expression of PD-L1 in tumor tissues and prognosis of patients with NSCLC. (A) PD-L1-positive and (B) PD-L1-negative IHC staining in tumor tissues of patients with NSCLC. (C) PD-L1-positive and (D) PD-L1-negative HE staining in tumor tissues from patients with NSCLC (Scale bar, 100 µm). (E) Kaplan-Meier overall survival probability analysis of PD-L1-positive and PD-L1-negative patients. HE, hematoxylin and eosin; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1.

Article Snippet: The cells were then incubated with CK-FITC (1:50, Cat No. FITC-66187, Proteintech Group, Inc.), CD45-phycoerythrin (1:100, Cat No. PE-65082, Proteintech, China) and PD-L1-Alexa Fluor 647 (1:50, Cat No. CL647-65082, Proteintech, China) antibodies at room temperature in the dark for 2 h, followed by washing with PBS and DAPI staining at room temperature for 10 min.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer

doi: 10.3892/etm.2024.12583

Figure Lengend Snippet: Quantification of CTCs in patients with NSCLC and association with prognosis. (A) Representative images of cells stained with DAPI, CK-FITC and CD45-PE to identify CTCs in peripheral circulation of patients with NSCLC by immunofluorescence staining. Scale bar, 10 µm. (B) Kaplan-Meier analysis of OS in ≤5 CTC and >5 CTC groups of PD-L1-positive and PD-L1-negative patients. Quantification of CTCs, E-CTCs and M-CTCs in peripheral circulation of (C) PD-L1-positive and PD-L1-negative groups and (D) stage I-II and stage III-IV groups. Data are presented as the mean ± SD. Kaplan-Meier analysis of survival probability in (E) The OS of M-CTC on the PD-L1 + -group and PD-L1 - -group; (F) The OS of E-CTC on the PD-L1 + -group and PD-L1 - -group. * P<0.05, *** P<0.001. CK, cytokeratin; CTC, circulating tumor cell; E-CTC, epithelial-type CTC; M-CTC, mesenchymal-type CTC; ns, not significant; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1; PE, phycoerythrin; WF, white field.

Article Snippet: The cells were then incubated with CK-FITC (1:50, Cat No. FITC-66187, Proteintech Group, Inc.), CD45-phycoerythrin (1:100, Cat No. PE-65082, Proteintech, China) and PD-L1-Alexa Fluor 647 (1:50, Cat No. CL647-65082, Proteintech, China) antibodies at room temperature in the dark for 2 h, followed by washing with PBS and DAPI staining at room temperature for 10 min.

Techniques: Staining, Immunofluorescence

Journal: Experimental and Therapeutic Medicine

Article Title: Detection of PD‑L1 expression and epithelial‑mesenchymal transition of circulating tumor cells in non‑small cell lung cancer

doi: 10.3892/etm.2024.12583

Figure Lengend Snippet: Quantification of PD-L1+ CTCs and CTC subtypes in patients with NSCLC. (A) Representative images of cells stained with DAPI, pcytokeratins (CK-FITC), CD45-PE and PD-L1 (AF647) to identify PD-L1 + CTCs in peripheral circulation of patients with NSCLC by immunofluorescence staining. Scale bar, 10 µm. (B) Kaplan-Meier analysis of survival probability in ≤5 PD-L1 + CTCs and >5 PD-L1 + CTCs groups of PD-L1-positive patients, and ≤5 CTCs and >5 CTCs groups of PD-L1-negative patients. Quantification of PD-L1 + CTCs, PD-L1 + E-CTCs and PD-L1 + M-CTCs in peripheral circulation of (C) PD-L1-positive and PD-L1-negative groups and (D) stage I-II and stage III-IV groups. Data are presented as the mean ± SD. Kaplan-Meier analysis of survival probability in (E) PD-L1 + M-CTC on the PD-L1 + -group and PD-L1 - -group; (F) OS of PD-L1 + E-CTC on the PD-L1 + -group and PD-L1 - -group. * P<0.05, *** P<0.001. CK, cytokeratin; CTC, circulating tumor cell; E-CTC, epithelial-type CTC; M-CTC, mesenchymal-type CTC; ns, not significant; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand 1; PE, phycoerythrin; WF, white field.

Article Snippet: The cells were then incubated with CK-FITC (1:50, Cat No. FITC-66187, Proteintech Group, Inc.), CD45-phycoerythrin (1:100, Cat No. PE-65082, Proteintech, China) and PD-L1-Alexa Fluor 647 (1:50, Cat No. CL647-65082, Proteintech, China) antibodies at room temperature in the dark for 2 h, followed by washing with PBS and DAPI staining at room temperature for 10 min.

Techniques: Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant

doi: 10.1038/s41467-024-47454-7

Figure Lengend Snippet: The 10-GE porcine xenografts were removed from the brain-dead human recipient three days after xenotransplantation, and single-cell RNA-sequencing was performed on FACS-enriched immune cells from the right xenograft using pig- and human-specific CD45+ antibodies. Data were aligned to the hybrid human-porcine reference genome. a&b) UMAP of sorted CD45+ cells ( n = 6513 cells) colored by cell type ( a ) and species ( b ). c , d Enumeration of sequenced human and porcine immune cells. e Expression of select marker genes in human and pig immune cell clusters.

Article Snippet: Cells were incubated in 5 μL per 100 μL cell suspension Human TruStain FcX, Fc Receptor blocking solution, (Biolegend Inc., Cat. No. 422302) for 10 minutes at room temperature and then stained with 5 μL per test of 0.5 mg per mL anti-human CD45 FITC clone 2D1 (Biolegend Inc., Cat. No. 368508) and 10 μL per 100 μL cell suspension of mouse anti-pig CD45-Alexa Fluor 647 conjugate antibody clone K252.1E4 (Bio-Rad Laboratories, Inc, Cat. No. MCA1222A647).

Techniques: RNA Sequencing, Expressing, Marker

Journal: Nature Communications

Article Title: Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant

doi: 10.1038/s41467-024-47454-7

Figure Lengend Snippet: Spatial transcriptomics was performed on serial needle core biopsies of 10-GE porcine kidneys before and after transplantation into a brain-dead human recipient. Biopsies were obtained from either the right (pre-transplant and day 3 samples) or left (day 1 and day 3T) xenografts. Cell type signatures were identified from reference transcriptomes using cell2location (for Pig T cells, top plots in 3a) or expression of individual marker genes (e.g. CD3E and CD19 ) for adaptive immune cell types that were not well represented in CD45+ immune cells sorted from the xenograft explants (see Fig. ) and were therefore not included in the reference transcriptomes passed to cell2location. a No detection of human T cell genes in xenograft biopsies at any time point (left) with quantification of gene expression levels (right). Pig T cells were readily detectable in the xenograft (top row). b Calculated total ( left ) and normalized ( right ) cell abundance for various pig T cells in the indicated biopsies. Note that cell abundance for human T cells was imputed from expression of hg38-CD3E , ss11-CD19 , and hg38-CD19 genes as shown ( a ). c No detection of human B cell genes in xenograft biopsies at any time point (left) with quantification of gene expression levels (right). Pre-tx pre-transplant. day 3T biopsy was taken on post-transplant day 3 at study termination.

Article Snippet: Cells were incubated in 5 μL per 100 μL cell suspension Human TruStain FcX, Fc Receptor blocking solution, (Biolegend Inc., Cat. No. 422302) for 10 minutes at room temperature and then stained with 5 μL per test of 0.5 mg per mL anti-human CD45 FITC clone 2D1 (Biolegend Inc., Cat. No. 368508) and 10 μL per 100 μL cell suspension of mouse anti-pig CD45-Alexa Fluor 647 conjugate antibody clone K252.1E4 (Bio-Rad Laboratories, Inc, Cat. No. MCA1222A647).

Techniques: Transplantation Assay, Expressing, Marker, Gene Expression

Journal: Nature Communications

Article Title: Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant

doi: 10.1038/s41467-024-47454-7

Figure Lengend Snippet: scRNA-seq was performed on CD45+ immune cells sorted from the right porcine kidney xenograft at explant (see Supplementary Fig. ), and macrophage clusters were selected for analysis. a Expression of M1 (red) and M2 (blue) genes in human and pig macrophages (see Supplementary Dataset 1 and ref. for full gene list). b Expanded view of top 50 most highly expressed M1 and M2 genes in each species. Note M2 > M1 genes for both species. c Composite gene expression score of pig and human macrophages of M1-like pro-inflammatory (red) and M2-like anti-inflammatory (blue) gene signatures . UMAPs were generated from re-clustering of macrophage clusters selected from Supplementary Fig. . d Expression of select anti- and pro-inflammatory cytokine genes in pig and human macrophages. Average gene expression is visualized such that the mean of the scaled expression dataset is set at 0 with a standard deviation of 1. ss11-IL6 was not detected.

Article Snippet: Cells were incubated in 5 μL per 100 μL cell suspension Human TruStain FcX, Fc Receptor blocking solution, (Biolegend Inc., Cat. No. 422302) for 10 minutes at room temperature and then stained with 5 μL per test of 0.5 mg per mL anti-human CD45 FITC clone 2D1 (Biolegend Inc., Cat. No. 368508) and 10 μL per 100 μL cell suspension of mouse anti-pig CD45-Alexa Fluor 647 conjugate antibody clone K252.1E4 (Bio-Rad Laboratories, Inc, Cat. No. MCA1222A647).

Techniques: Expressing, Gene Expression, Generated, Standard Deviation