Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 1. Sulfatide induces surface CXCR4 up-regulation on human blood leukocytes. Cells were isolated from peripheral blood, incubated with sulfatide (100 lg/mL) for 1 h at 37C and stained for CXCR4. (A) Flow cytometry histogram of a representative experiment. The dotted line, solid line and grey area represent respectively the isotype control, CXCR4 baseline level, and CXCR4 level after sulfatide treatment. (B, C) Summarized results of multiple experiments. Data is presented as mean fluorescence intensity relative to the basal expression (white and black columns represent respectively the control and sulfatide treatment while bars indicate SEM, n = 4). * p value <0.05, ** p value <0.01.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Incubation, Staining, Flow Cytometry, Control, Fluorescence, Expressing

Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 3. Sulfatide-induced CXCR4 up-regulation is critically mediated by L-selectin in CD4+ T cells. (A) L-Selectin was cross-linked with Mel-14 and secondary antibodies; then, CXCR4 surface levels were measured (white and black columns represent the control and cross-linking treatments, respectively, while bars indicate SEM; n = 3). L-Selectin cross-linking induced CXCR4 up-regulation of murine spleen subsets. K562 cells were incubated with sulfatide (B) or Lam1-116 cross-linking antibody (C) (white and black columns represent the control and treatment groups, respectively; the striped column represents transfected CXCR4 up- regulation as measured by anti-HA tag; the bars indicate SEM; n = 3). Surface CXCR4 expression on K562 cells was up-regulated by sulfatide or Lam1-116 only in the presence of WT L-selectin (WT) and not in the presence of a mutant (~16) that lacks 16 of 17 intracellular amino acids, or the parent cells which do not have L-selectin. (D) Galactosylcerebroside (GalCer) has the same structure as sulfatide, but differs only by a sulfate group, as marked by arrow. (E) Galactosylcerebroside had no effects on CXCR4 up-regulation of CD4+ T cells when compared to no treatment (NT) (n = 3). (F) Splenocyte subsets were isolated from WT and L-sel–/– animals, incubated with sulfatide, then stained for CXCR4. The dotted line, solid line and grey area represent respectively the isotype control, CXCR4 baseline level, and CXCR4 level after sulfatide treatment. The histogram is representative of multiple experiments. Sulfatide induced CXCR4 up-regulation on CD8+ T cells and B cells independently of L-selectin, while it was essential on CD4+ and CD4+CD25+ T cells (n = 3). Galactosylcerebroside had no effects on any cells.* p value <0.05.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Control, Incubation, Transfection, Expressing, Mutagenesis, Isolation, Staining

Journal: European journal of immunology

Article Title: Up-regulation of leukocyte CXCR4 expression by sulfatide: an L-selectin-dependent pathway on CD4+ T cells.

doi: 10.1002/eji.200737118

Figure Lengend Snippet: Figure 5. Sulfatide-induced CXCR4 up-regulation has signaling and functional consequences for CD4+ T cells. (A) CD4+ T cells were incubated with sulfatide for various times and stained for CXCR4. CXCR4 was up-regulated rapidly; 50% of the maximal up- regulation was reached as early as after 2 min while galactosylcerebroside had no effect at any time point. (B, C) CD4+ T cells were incubated with sulfatide (S) or galactosylcerebroside (G), then stimulated with CXCL12 (200 ng/mL) for 30 s. Western blot analysis showed increased CXCL12-induced tyrosine phosphorylation (B) and PI3K activation (C) after exposure to sulfatide. Blots were quantified and expressed as relative intensity (n = 3). (D) CD4+ T cells were incubated with sulfatide or galactosylcerebroside (GalGer) and loaded into the top chamber of a Transwell plate with either CXCL12 (black) or CCL5 (stripes) in the bottom chamber for 2 h. Migration to CXCL12 was enhanced by exposure to sulfatide but not galactosylcerebroside or no treatment (NT). The migration to CCL5 or to no chemokine (white) was not affected. * p value <0.05.

Article Snippet: PE-conjugated isotype control and PE- and FITC-conjugated anti-CXCR4 (12G5) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Functional Assay, Incubation, Staining, Western Blot, Phospho-proteomics, Activation Assay, Migration

Journal: Cell

Article Title: Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells

doi: 10.1016/j.cell.2022.04.039

Figure Lengend Snippet:

Article Snippet: Anti-human CD184 , Miltenyi Biotec , Cat# 130-117-519; RRID:AB_2734059.

Techniques: Blocking Assay, Plasmid Preparation, Recombinant, Electroporation, CRISPR, Adjuvant, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Inflammation induces pro-NETotic neutrophils via TNFR2 signaling

doi: 10.1101/2021.06.21.448937

Figure Lengend Snippet: A-B) Total CCR5 expression was detected in whole cell lysates from rPMN and cPMN by western blot analysis. GAPDH immunostaining served as loading control. The blot in A is representative for three independent experiments. A quantification of the CCR5 expression as ratio between CCR5- and GAPDH-derived signals is depicted in B (SEM; n=3; t-test; n.s.=not significant). B) CCR5 was detected on rPMN (left) and cPMN (right) by surface immunostaining (upper part) or by total immunostaining (lower part) using 0.1% saponin to permeabilize the cells. Shown are representative flow cytometry histograms taken from three independent experiments. C) rPMN and cPMN were adhered on coverslips, fixed, permeabilized and immunostained for CD66b (blue) and CCR5 (red). In addition F-actin (phalloidin-AF488) and nuclei (DAPI) were labelled. Images of 28 cells from three independent experiments were acquired using structured-illumination microscopy (3D-SIM). D) rPMN and cPMN were fixed and stained with phalloidin-AF488 to quantify F-actin by imaging flow cytometry. Shown is the total intensity from 6 independent experiments (n=6, SEM, ANOVA, *p<0.05, **p<0.01). E) PMN were stimulated with GM-CSF/ IFNγ in the presence or absence of either CytoD (250nM) or Jas (500nM) for one day. The expression of CCR5 was quantified using flow cytometry (n=3, SEM, ANOVA, *p<0.05, **p<0.01). G-H) The expression of PMN lineage-associated receptors ( G) , CD66b, CD15, CD62L, CD16, HLA-DR and CD49d) as well as chemokine receptors ( H) , CXCR1, CXCR3, CXCR4, CCR3, CCR4) were analyzed on rPMN, CCR5 − and CCR5 + cPMN by flow cytometry. The cells were gated for CD66b + (rPMN) and their expression of CCR5 (CD66b + cPMN). The histograms are representative for three independent experiments.

Article Snippet: The following primary antibodies were employed: CCR5-PE (Miltenyi, Bergisch Gladbach, Germany); CCR5 unlabeled; CCR1-APC-Fire 750, CXCR1-APC, CCR4-BV605, CD66b-PerCP-Cy5.5, CD15-AF594, CD62L-FITC, CD49d-APC, CD16-APC-Cy7, HLA-DR-AF700, TNFα-PE-Dazzle594, TNFR1-APC, TNFR2-PE-Cy7 (Biolegend, San Diego, USA); CXCR3-APC, CXCR3-PE-Cy5, CXCR4-PE-Cy5, CCR3-PE CCR3-PE-CF594, CD15-BV605, CD49d-PE-Cy5 (BD Bioscience, Heidelberg, Germany); ELANE (MerckMillipore, Darmstadt, Germany); CXCR4-FITC (R&D Systems, Minneapolis, USA); TNFα (purified), CCR5 (purified) (R&D Systems, Minneapolis, USA); azurocidin (Abcam, Cambridge, UK); Anti-mouse-Cy5 (Dianova, Hamburg, Germany).

Techniques: Expressing, Western Blot, Immunostaining, Control, Derivative Assay, Flow Cytometry, Microscopy, Staining, Imaging

Journal: Stem Cell Research & Therapy

Article Title: Allogeneic human umbilical cord Wharton’s jelly stem cells increase several-fold the expansion of human cord blood CD34+ cells both in vitro and in vivo

doi: 10.1186/s13287-020-02048-0

Figure Lengend Snippet: Cell adhesion markers and migration of UCB CD34+ cells expanded with hWJSCs and hWJSC-CM. A Note the percentage of the most primitive CD34+CXCR4+ cells in the hWJSCs and hWJSC-CM-expanded groups was closest to the initial seeding cell numbers on day 0 (dotted line) compared to the controls. B Note significantly greater percentage of N-cadherin+ cells after expansion of CD34+ cells with hWJSCs as compared to controls. C (a–c) Phase-contrast images of the bottom chamber of transwell migration assays. Note increased number of CD34+ cells that have migrated from the top to the bottom chamber when cultured in the presence of hWJSCs and hWJSC-CM compared to controls. (d–f) There was an increased number of N-cadherin positive cells among the migrated cells expanded with hWJSCs and hWJSC-CM compared to controls. D Note significantly greater viable CD34+ cell numbers (MTS assay) lying in the bottom chamber of transwell migration chambers after expansion with hWJSCs as compared to controls. All values are expressed as mean ± SEM of 3 biological samples with 3 replicates for each sample. * p < 0.05

Article Snippet: The cell pellets from experimental and control groups were collected and incubated with conjugated anti-human CD34-APC and CXCR4-FITC antibodies (1:10) (Mitenyi Biotec) for 30 min on ice.

Techniques: Migration, Cell Culture, MTS Assay