rat monoclonal anti cxcr4 igg antibody  (R&D Systems)

Journal: bioRxiv

Article Title: A Modular Layer-by-Layer Nanoparticle Platform for Hematopoietic Progenitor and Stem Cell Targeting

doi: 10.1101/2024.10.30.621186

Figure Lengend Snippet: ( A ) Median fluorescence intensity (MFI) values for all Ab–LbL NP groups after 1.5 h of incubation in human CD34+ cells, normalized to PAA NPs. ( B ) Expression of surface receptors for cKit, CXCR4, CD90, CD105 on NALM6 cells, Toledo cells, and Raji cells. ( C ) Median fluorescence intensity (MFI) values after 1.5 h of incubation for anti-human Ab–PAA NPs, normalized to PAA NPs, in NALM6 cells, Raji cells, and Toledo cells. Statistical analysis is a one-way ANOVA with Tukey correction (**p<0.01, ***p<0.005, ****p<0.001). Bars represent mean with standard deviation. For (A) each shape represents a different donor (4 donors, 1-3 technical replicates). For (C) each dot is one technical replicate (2 biological replicates with 3 technical replicates each).

Article Snippet: Antibody rat-anti-mouse-CXCR4 (CD184, clone 2B11), rat-anti-mouse CD105 (clone MJ7/18), mouse-anti-human CD45 (clone HI30), mouse-anti-human CXCR4 (CD184, clone 12G5), mouse-anti-human CD90 (Thy-1, clone 5E10), and mouse-anti-human CD105 (clone SN6) were purchased from Thermo Fisher Scientific (Massachusetts, US).

Techniques: Fluorescence, Incubation, Expressing, Standard Deviation

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: Glutathione S-Transferase P Influences Redox and Migration Pathways in Bone Marrow

doi: 10.1371/journal.pone.0107478

Figure Lengend Snippet: (A) Migration of BMDDCs to CXCL12. Wide type and Gstp1/p2 −/− BMDDCs were either left untreated (control) or stimulated with 200 ng/ml CXCL12 for 16 h. Values are average percentages of migration (± SD ) from six independent experiments with asterisks (*) indicating statistical significant differences between WT and Gstp1/p2 −/− BMDDCs ( p <0.05). ( B, C ) Intracellular calcium (B); plasma membrane potential dynamics (C) in WT and Gstp1/p2 −/− BMDDCs in response to CXCL12. Arrows indicate the addition of CXCL12. Data are representative traces of three independent experiments. ( D ) Kinetics of Akt and ERK phosphorylation in BMDDCs with response to CXCL12. Wild type and Gstp1/p2 −/− BMDDCs were serum starved for 2 h. 200 ng/ml CXCL12 was then added and cells were collected at various time points. Representative immunoblots show phosphorylation of Akt and ERK in WT and Gstp1/p2 −/− BMDDCs after stimulation with CXCL12 for the indicated periods. p-Akt or p-ERK levels were quantified and normalized relative to total Akt or ERK protein for each time point. The ratio (p-Akt:total Akt or p-ERK:total ERK) at 0 min CXCL12 exposure was assigned a value of one and all other ratios are shown relative to this value. Bars represent the means (± SD ) from three independent experiments. ( E, F ) Impact of GSTP ablation on CXCR4, IP3R and SHP-2 levels in BMDDCs. Total protein levels were evaluated by immunoblots. Actin served as a loading control. Surface expression of CXCR4 was further analyzed by flow cytometry. Representative histograms are shown: CXCR4 expression in WT and Gstp1/p2 −/− BMDDCs compared to isotype control showing the mean fluorescence intensity (MFI) and statistical summary of three independent measurements presented as means (± SD ) with asterisks (*) indicating statistically significant differences between WT and Gstp1/p2 −/− BMDDCs (p<0.05). ( E ). Relative gene expression levels were quantified by Real-Time RT-PCR. Bars represent the means (±SD ) from three independent experiments ( F ). ( G ) Effect of SHP-2 specific inhibitor PHPS1 and CXCR4 specific antagonist AMD3100 on CXCL12-stimulated chemotaxis of BMDDCs. WT and Gstp1/p2 −/− BMDDCs were either untreated (control) or pretreated with 10 µM PHPS1 or 1 µM AMD3100 for 2 h, and then cells were either left untreated or stimulated with 200 ng/ml CXCL12 for 6 h. Values are average percentages of specific chemotaxis (percentage of cells migrating to medium alone was subtracted from the percentage of cells migrating to medium with CXCL12) (± SD ) from three independent experiments, with asterisks (*) indicating statistically significant differences between control and drug treatments ( p <0.05). Chemotaxis of WT cells in the absence of inhibitor is considered 100% migration.

Article Snippet: Cell surface CXCR4 expression was determined by fluorescence-activated cell sorter (FACS) analysis using FITC-conjugated rat anti-mouse CXCR4 mAb or FITC-conjugated rat IgG2b (both from BD Pharmingen) as isotype control.

Techniques: Migration, Western Blot, Expressing, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Chemotaxis Assay

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: bioRxiv

Article Title: Loss of LRP1 in adult neural stem cells impairs migration to ischemic lesions

doi: 10.1101/2022.08.17.504194

Figure Lengend Snippet: Representative images are shown from SVZs in (A, C, E, G) Control (Cntrl) and (B, D, F, H) LRP1-KO mice 2-weeks after MCAO of (A,B) tdTomato+ cells, (C,D) CXCR4 immunostained cells, (E, F) Channels were merged to show tdTomato (red), CXCR4 (green) and nuclear DAPI (blue). Areas in the white box are magnified in (G, H) to show orthogonal views. (I) Quantification of CXCR4 intensity in tdTomato+ cells (n=5-6 mice/group). (J) qRT-PCR of CXCR4 was in tdTomato+ sorted cells (n=4). Results are averages ± SEM. Significant differences were tested using ANOVA followed by Tukey’s HSD and are represented on the graph.

Article Snippet: Antibodies used were rat anti-CXCR4 (R&D MAB21651 1:25); chicken anti-GFAP (Sigma AB5541 1:500); donkey anti-chicken Alexa 488 (Jackson 703-545-155 1:200); donkey anti-rat Alexa 488 (Jackson 711-545-152 1:200).

Techniques: Quantitative RT-PCR