phycoerythrin pe conjugated rat anti mouse cxcr4 antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Characterization of the mesendoderm progenitors in the gastrulating mouse embryo

doi: 10.1101/2024.04.28.591221

Figure Lengend Snippet: A. EpiSC lines of Mixl1WT and Mixl1KO genotypes, and harboring FLAG-Mixl1 in HPRT locus that can be activated by induction with Doxycycline. Gene editing of Mixl1 locus in Mixl1 KO and Mixl1 Dox inducible cell line. B. The expression of Mixl1 in the cell lines induced by Doxycycline treatment on Day 0, 1, 2 and 3 (D0DOX, D1DOX, D2 DOX and D3 DOX respectively).The panel highlights (grey) the time when expression peaks for individual Dox induction timings. C. Principal component analysis of gene expression profile assayed by RNA sequencing of the samples-D1Dox (Day 2), NoDox(Day 2), D0Dox (Day4), NoDox (Day 4). D. Heat map presentation of Mixl1 responsive genes upregulated with Day 1 (early) and Day 3 (late) induction of Mixl1 activity in comparison to NoDox treatment. Gene Ontology enrichment analysis of the upregulated genes with Day 1 versus Day 3. E. Flow cytometry analysis of CXCR4+ cells in the differentiating EpiSCs with different timing of Mixl1 induction (N=3). The left panel is all CXCR4+ cells as a percentage of total cells. The right panel includes cells that were gated as highly positive for CXCR4. F. Expression of Sox17 transcript in MixL1WT, MixL1KO or MixL1Dox mouse EpiSCs with either No Dox treatment or MixL1 induction via Doxycycline at Day 0 (D0Dox), 1 (D1Dox), 2 (D2Dox) and 3 (D3Dox) of differentiation.

Article Snippet: CXCR4 (CD184) staining for endoderm propensity quantification was performed using phycoerythrin (PE)-conjugated rat anti-mouse CXCR4 antibody (R&D Systems) in Dulbecco’s phosphate-buffered saline with 10% Fetal Calf Serum (Flow cytometry buffer) for 45 minutes at room temperature and washed thrice in Flow cytometry buffer.

Techniques: Expressing, RNA Sequencing Assay, Activity Assay, Comparison, Flow Cytometry

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A–B) Immunohistochemistry revealed, that in normal human skin, CXCR4 is mainly expressed in the epidermis. In psoriatic skin, CXCR4 is also expressed on a big number of infiltrating cells. Quantitative image analysis showed that significantly more CXCR4 + cells were present in the dermis of psoriatic skin lesions (n = 8) than in normal human skin (n = 8). Staining with isotype matched control IgG confirmed the specificity of the CXCR4 staining. Scale bar represents 100 μm. *** P<0.001. (C) Real-time RT-PCR analysis of RNA obtained from whole ear skin extracts of inflamed K14-VEGF-A transgenic mice (day 21 after oxazolone challenge), untreated K14-VEGF-A transgenic, and wild-type mice (n = 5 per group). The expression of CXCR4 and SDF-1 was significantly up-regulated in uninflamed K14-VEGF-A transgenic mouse skin compared to wild-type mice and was further increased in the inflamed skin of K14-VEGF-A transgenic mice. The expression of CXCR7 was slightly lower in the skin of K14-VEGF-A transgenic mice. (D–E) Immunofluorescence stains for CXCR4 revealed that inflamed K14-VEGF-A transgenic mice have a significantly increased CXCR4 + tissue area as compared with uninflamed K14-VEGF-A transgenic mice and wild-type mice. Scale bar represents 100 μm. Data represent mean ± SD. * P<0.05; *** P<0.001. ns, not significant.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Transgenic Assay, Expressing, Immunofluorescence

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A) Hemizygous K14-VEGF-A transgenic mice (n = 20) were sensitized with 2% oxazolone on day -5 and challenged on day 0 by topical application of 1% oxazolone on the ears. Starting on day 7, mice received s.c injections of AMD3100 (CXCR4-antagonist) or PBS (vehicle) (n = 10 per group) every 12 hours. Two independent experiments were performed. (B) Treatment with AMD3100 (△) reduced ear swelling, as compared with PBS-treated controls (□) Data represent mean±SEM. (C) Reduced weight of ear draining LN after 14 days of AMD3100 treatment (day 21), as compared with PBS-injected mice. Data represent mean±SD. *** P<0.001. (D-G) AMD3100-treated animals showed reduced ear redness on day 10 (E) and day 21 (G) as compared to PBS-treated animals (D,F).

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Transgenic Assay, Injection

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A–B) Representative fluorescent images of MECA-32 + blood vessels (red) and LYVE-1 + lymphatic vessels (green) in the inflamed ear skin of PBS (A) and AMD3100-treated (B) K14-VEGF-A transgenic mice. One ear half is shown. Scale bar represents 100 μm. (C) Quantitative image analysis of MECA-32 + blood vessels (BV) revealed significantly reduced numbers of blood vessels per millimeter basement membrane (BM) in the inflamed ear skin of AMD3100-treated mice, as compared with PBS-treated control mice. (D–E) FACS analysis of CXCR4 expression by dermal blood vascular endothelial cells (BEC; D) and lymphatic endothelial cells (LEC; E) revealed that CXCR4 is expressed by BEC but not by LEC in vitro. (F) Immunofluorescence staining for CXCR4 (red), von Willebrand factor (VwF, green) and Podoplanin (Podo, purple) of inflamed ear skin (confocal image) demonstrates specific CXCR4 expression by blood vessels (BV) but not lymphatic vessels (LV). Scale bar represents 20 μm. Data represent mean ±SD. * P<0.05.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Transgenic Assay, Expressing, In Vitro, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A) On day -5, hemizygous K14 VEGF-A transgenic mice (n = 14) were sensitized with 2% oxazolone and challenged on day 0 with 1% oxazolone on the ears. Starting 7 days after challenge, mice received i.v injections of anti-SDF-1 antibody or isotype-matched IgG every second day. (B) Neutralization of SDF-1 (△) significantly reduced inflammatory ear swelling as compared with IgG-injected mice (□). Data represent mean ±SEM. (C-D) H&E stains of mouse ear sections at day 21 showed reduced edema and inflammatory cell infiltration in the anti-SDF-1 treated mice (D) in comparison to the IgG control group (C). One ear half is shown. Scale bar represents 100 μm. (E) In anti-SDF-1 treated mice, the weight of ear draining LNs was significantly reduced compared with controls. (F-H) Immunofluorescence staining for CD68 revealed a significant reduction in the percentage of area covered by macrophages in anti-SDF-1-treated animals as compared to IgG treatment. (I-L) Neutralization of SDF-1 decreased the size and numbers of blood vessels in the inflamed ear skin. (M) FACS analysis of CD11b + splenocytes revealed a clear expression of CXCR4 on their surface. (N) SDF-1 promoted the chemotactic migration of CD11b + splenocytes in vitro. (O-P) The chemotactic effect of SDF-1 was blocked by incubation with AMD3100 (O) or with an anti-SDF-1 antibody (P), but not with control IgG. Two independent experiments were performed. Data represent mean±SD. ** P<0.01; *** P<0.001.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Transgenic Assay, Neutralization, Injection, Immunofluorescence, Staining, Expressing, Migration, In Vitro, Incubation

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A–B) H&E stains of ear skin sections at day 21 showed that AMD3100 treatment reduced edema formation, epidermal thickening and inflammatory cell infiltration. (C–D) CXCR4 inhibition reduced the number of intraepidermal BrdU + proliferating cells in the inflamed ear skin. (E–H) The hyperproliferation-associated keratin 6 and loricrin, a marker of terminal epidermal differentiation, were less broadly expressed in the epidermis of AMD3100-treated mice than in PBS-treated mice. (I–L) Immunofluorescence staining of the two macrophage markers F4/80 and CD68 revealed a significant reduction in the percentage of area covered by macrophages in AMD3100-treated mice compared to PBS treatment. (M–N) Inhibition of CXCR4 decreased the number of intraepidermal CD8 + T-cells in the inflamed ear skin. One ear half is shown. (O–P) Computer-assesed quantification of epidermal thickness (O), number of intraepidermal BrdU + cells (P), the percentage of covered area by F4/80 (Q) and CD68 (R) postitive macrophages and the number of intraepidermal CD8 + cells (S). Scale bars represent 100 μm. Data represent mean ±SD. * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Inhibition, Marker, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A, B) Real-time RT-PCR analysis of extracts of IMQ-inflamed ear skin (8 consecutive days) and non-inflamed skin (n = 5 per group). The expression of CXCR4 and SDF-1 was significantly upregulated in IMQ-treated ear skin compared to uninflamed skin. (C–E) Immunofluorescence stains of ear skin for CXCR4 revealed a significantly increased CXCR4 + tissue area in IMQ-treated mice as compared with uninflamed skin. (F) Mice (n = 5 per group) received AMD3100 or PBS injections every 12 hours. 12 hours after the first injection, IMQ was applied topically, followed by daily applications for 8 days. (G) Treatment with AMD3100 (△) significantly reduced ear swelling as compared with PBS-treated controls (□). (H–J) H&E stains of ear skin sections showed that AMD3100 treatment (I) significantly reduced epidermal thickening compared to PBS-treated mice (H). Scale bar represents 100 μm. (K) CXCR4 inhibition significantly reduced the number of intraepidermal BrdU + proliferating cells in the inflamed ear skin, as compared with control mice. Data represent mean±SD. * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Injection, Inhibition

Journal: PLoS ONE

Article Title: An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

doi: 10.1371/journal.pone.0093665

Figure Lengend Snippet: (A–H) Representative images of immunofluorescence stains for CD68 + (A, B) and F4/80 + (C, D) macrophages, MHCII + antigen presenting cells (E, F) and MECA-32 + blood vessels (G, H) in the IMQ-inflamed ear skin of PBS and AMD3100-treated mice. One ear half is shown. Scale bars represent 100 μm. (I–K) Quantitative image analysis showed a significant reduction in the percentage of area covered by macrophages (I, J) and antigen presenting cells (K) in AMD3100-treated mice. (L) Inhibition of CXCR4 decreased the number of Meca-32 + blood vessels (BV). (M–P) Real-time RT-PCR analyses of RNA from whole ear skin extracts of imiquimod-inflamed mice showed a significant downregulation of CXCL3 (M), CCL20 (N), S100a7 (O) and S100a8 (P). Data represent mean ±SD. * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: A single cell suspension of CD11b + splenocytes was stained at 4°C for 30 minutes with PE-labeled rat anti-mouse CXCR4 antibody (eBioscience) or isotype control antibody (BioLegend).

Techniques: Immunofluorescence, Inhibition, Quantitative RT-PCR

Journal: Pharmaceutics

Article Title: Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium

doi: 10.3390/pharmaceutics14061194

Figure Lengend Snippet: Analysis of CXCR4 and PSGL-1 expressions on the surfaces of EPCs after transfection with CXCR4 and PSGL-1 mRNA. A total of 1 × 10 5 EPCs were seeded and transfected after 24 h with either 1 µg CXCR4, 1 µg PSGL-1 mRNA, or both mRNAs (mRNA cocktail). The CXCR4 and PSGL-1 expressions on the cell surfaces were analyzed 24 h post-transfection using flow cytometry. Cells treated only with medium and transfection reagent (TR) were used as negative control. Results are shown as mean + SD ( n = 3). Statistical differences were determined using one-way ANOVA multiple comparisons, followed by Bonferroni’s multiple comparisons test (** p < 0.01 and *** p < 0.001; ns: nonsignificant).

Article Snippet: Therefore, 1 × 10 5 transfected EPCs were detached, washed twice with 4% BSA/DPBS, and incubated with either PE rat anti-mouse CD162 (PSGL-1) antibody (BD Pharmingen, Heidelberg, Germany) or PE rat anti-mouse CXCR4 antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at RT.

Techniques: Transfection, Flow Cytometry, Negative Control

Journal: Pharmaceutics

Article Title: Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium

doi: 10.3390/pharmaceutics14061194

Figure Lengend Snippet: Analysis of cell viability of EPCs after transfection with CXCR4 and PSGL-1 mRNA. A total of 1 × 10 5 cells were seeded and transfected after 24 h with 1 µg of CXCR4, PSGL-1 mRNA, or both mRNAs (mRNA cocktail). The viability was analyzed 24 h post-transfection using a PrestoBlue™ cell viability assay. The viability of cells incubated only with the medium was set to 100%. Results are shown as mean + SEM ( n = 3).

Article Snippet: Therefore, 1 × 10 5 transfected EPCs were detached, washed twice with 4% BSA/DPBS, and incubated with either PE rat anti-mouse CD162 (PSGL-1) antibody (BD Pharmingen, Heidelberg, Germany) or PE rat anti-mouse CXCR4 antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at RT.

Techniques: Transfection, Viability Assay, Incubation

Journal: Pharmaceutics

Article Title: Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium

doi: 10.3390/pharmaceutics14061194

Figure Lengend Snippet: Chemotactic migration of mRNA-modified EPCs. A total of 1 × 10 5 EPCs were transfected with either 1 µg CXCR4 mRNA or with an mRNA cocktail containing 1 µg CXCR4 and 1 µg PSGL-1 mRNA. After overnight cultivation, 5 × 10 4 EPCs were seeded in 8 µm transwell inserts to analyze their migration capacity towards 50 ng/mL SDF-1α in a serum-reduced medium. Chemotactic migration was analyzed after 6 h at 37 °C by counting DAPI-stained migrated cells using ImageJ software. As controls, EPCs incubated with medium and medium containing transfection reagent (TR) were used. Scale bars represent 200 µm. Results are shown as mean + SD ( n = 4). Statistical differences were determined using one-way ANOVA multiple comparisons, followed by Bonferroni´s multiple comparisons test (**** p < 0.0001).

Article Snippet: Therefore, 1 × 10 5 transfected EPCs were detached, washed twice with 4% BSA/DPBS, and incubated with either PE rat anti-mouse CD162 (PSGL-1) antibody (BD Pharmingen, Heidelberg, Germany) or PE rat anti-mouse CXCR4 antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at RT.

Techniques: Migration, Modification, Transfection, Staining, Software, Incubation

Journal: Pharmaceutics

Article Title: Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium

doi: 10.3390/pharmaceutics14061194

Figure Lengend Snippet: Analysis of the adhesion strength of EPCs to activated endothelium using single-cell atomic force microscopy (AFM). Representative histograms of the adhesion signature of CXCR4 and PSGL-1 mRNA (mRNA cocktail) modified and native EPCs to TNF-α-activated and nonactivated endothelium and the quantification of adhesion forces are shown. Bonds formed between the cell surface ligands (PSGL-1) and molecules on HUVECs (E-selectin) broke slightly with increasing force until the cell completely detached from the HUVEC. The maximum downward force of the cantilever’s tip is referred to as detachment force (F detach ). ( n = 4). Statistical analysis was performed using two-way ANOVA, followed by Bonferroni´s multiple comparisons test (** p < 0.01 and **** p < 0.0001).

Article Snippet: Therefore, 1 × 10 5 transfected EPCs were detached, washed twice with 4% BSA/DPBS, and incubated with either PE rat anti-mouse CD162 (PSGL-1) antibody (BD Pharmingen, Heidelberg, Germany) or PE rat anti-mouse CXCR4 antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at RT.

Techniques: Microscopy, Modification

Journal: Pharmaceutics

Article Title: Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium

doi: 10.3390/pharmaceutics14061194

Figure Lengend Snippet: Dynamic adhesion of mRNA-modified EPCs to activated endothelium. ( A ) HUVECs were treated with 10 ng/mL TNF-α, and E-selectin expression was analyzed by flow cytometry to analyze the successful activation of the cells. ( n = 3). Statistical analysis was performed using a t -test (* p < 0.05 and *** p < 0.001). ( B ) EPCs were transfected with an mRNA cocktail of CXCR4 and PSGL-1 mRNA (1 µg each). After 24 h, the cells were stained with PKH26 and perfused over TNF-α-activated HUVECs in a flow chamber at 0.11 mL/min and a shear stress of 0.1 dyn/m 2 . ( B ) Representative images of rolling EPCs are shown every 2 s. Red arrows indicate a slowly rolling cell, and yellow arrows point to a fast-moving cell not interacting with the activated HUVECs. ( C ) Representative images of recordings at 1, 3, 5, and 7 min after starting the flow. ( D ) The number of EPCs adhered to HUVECs after 7 min was quantified ( n = 3). Statistical analysis was performed using two-way ANOVA, followed by Bonferroni´s multiple comparisons test (** p < 0.01).

Article Snippet: Therefore, 1 × 10 5 transfected EPCs were detached, washed twice with 4% BSA/DPBS, and incubated with either PE rat anti-mouse CD162 (PSGL-1) antibody (BD Pharmingen, Heidelberg, Germany) or PE rat anti-mouse CXCR4 antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at RT.

Techniques: Modification, Expressing, Flow Cytometry, Activation Assay, Transfection, Staining, Shear

Journal: Stem Cell Research & Therapy

Article Title: The combination of G-CSF and AMD3100 mobilizes bone marrow-derived stem cells to protect against cisplatin-induced acute kidney injury in mice

doi: 10.1186/s13287-021-02268-y

Figure Lengend Snippet: Effects of G-CSF/AMD3100 on the mobilization of stem cells. Four days after cisplatin injection, the peripheral blood of the mice was obtained and analysed by a flow cytometer. a CXCR4 + cells. b CXCR4 + CD34 + cells. c CXCR4 + CD133 + cells. Data represent the mean ± SD ( n = 6 per group). * P < 0.05, ** P < 0.01, and vs. the cisplatin group and the indicated test group

Article Snippet: The remaining cells were labelled with FITC-labelled rat anti-mouse CD34 (diluted 1:100; BD Biosciences, #560238), APC-labelled rat anti-mouse CD133 (diluted 1:100; BioLegend, #141207), FITC-labelled rat anti-mouse CD44 (diluted 1:100; BD Biosciences, #553133), PE-labelled rat anti-mouse CXCR4 (diluted 1:200; BD Biosciences, #561734), and PE-labelled rat anti-mouse C-kit (diluted 1:100; BD Biosciences, #553355).

Techniques: Injection, Flow Cytometry