Journal: PLoS ONE

Article Title: WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility

doi: 10.1371/journal.pone.0070364

Figure Lengend Snippet: a Chemotactic response of freshly isolated WIP +/+ and WIP −/− B cells to CXCL13 (400 nM) in Boyden chambers. b Polarization and migration frequencies of WIP +/+ and WIP −/− B cells settled on ICAM-1-containing planar membranes coated with CXCL13. In ( a ) and ( b ) each dot corresponds to one experiment; black thick bar, averaged value. c Mean speed values (left panel) and Straightness index (right panel) of WIP +/+ and WIP −/− B cells in the same conditions than in ( b ); each dot corresponds to a single cell. d Tracks of representative WIP +/+ and WIP −/− B cells migrating on the planar membranes; each line corresponds to a single cell track. e DIC and F-actin images of representative WIP +/+ (left panels) and WIP −/− (middle panel) B cells; right panel, lamellipodium frequency in control and deficient B cells. Data on ( c ) and ( e ) is the merge of three experiments. *, p<0.05; **, p<0.001; ***, p<0.0001.

Article Snippet: The recombinant murine CXCL13 chemokine (Peprotech) was added at the indicated concentrations to the lower chamber filled with 600 μls of RPMI 10% FCS.

Techniques: Isolation, Migration, Control

Journal: PLoS ONE

Article Title: WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility

doi: 10.1371/journal.pone.0070364

Figure Lengend Snippet: − / − B cells. Purified WIP +/+ and WIP −/− B cells were transduced with recombinant lentivirus expressing GFP (GFP) or full-length WIP-GFP (WIP) as described in Materials and Methods; 24 h later, they were used for the different assays. a Total lysates of the indicated transduced B cells were used to detect the expression of the WIP construct by western-blot with anti-GFP. b Migration frequency of the indicated transduced B cells in response to 400 nM CXCL13 in Boyden chambers; data merged from two experiments are shown. c Mean speed values, d F-actin-rich lamellipodium frequency, and e representative tracks of the specified transduced B cells migrating on the planar membranes; each dot is a single cell in ( c ); data from a representative experiment are shown in ( e ). *, p<0.05; **, p<0.001.

Article Snippet: The recombinant murine CXCL13 chemokine (Peprotech) was added at the indicated concentrations to the lower chamber filled with 600 μls of RPMI 10% FCS.

Techniques: Purification, Transduction, Recombinant, Expressing, Construct, Western Blot, Migration

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

doi: 10.1016/j.omtm.2016.11.001

Figure Lengend Snippet: Reconstitution of Immune Cell Functions following LV Gene Therapy in Primary Recipients Assays of immune cell function 16 weeks after BM transplantation. Bars show the mean ± SD. (A) The percentage of cells that underwent ≥1 cell division 72 hr after incubation with CD3/CD28 antibodies or P/I (as measured by CellTrace Violet dilution). Shown are cells within CD4 (left) or CD8 (right) staining gates by flow cytometry. Data are from one of the three independent experiments: n = 3 (KO and WT mock, MND.hWASp) and 5 (650.MND.hWASp). (B) Flow cytometry analysis showing CellTrace Violet labeled splenocytes 72 hr post-CD3/CD28 stimulation, gated on live and either CD4 + or CD8 + populations. Numbers indicate the percentages that have proliferated after CellTrace Violet labeling. (C) B cell (CD43 − splenocyte) migration in response to media only ( − ) or media supplemented with CXCL13 chemokine ( + ). Each dot indicates the percentage of B cells from a single mouse that migrated through the 5-μm-pore transwell. Data are from two independent experiments: n = 5 (KO mock, 650.MND.hWASp), 3 (MND.hWASp), and 8 (WT mock). (D) IgG and IgM and (E) anti-double-stranded DNA antibody levels in the serum of primary recipients as determined by ELISA 16 weeks post-transplant. Data are from three independent experiments: n = 5 (KO and WT mock), 4 (MND.hWASp), 2 (WT) (D only), and 12 (650.MND.hWASp). Serum from an autoimmune WAS chimera (E only) with high serum anti-DNA antibodies was used as a positive control. The p value was determined by unpaired two-tailed t test. *p < 0.024.

Article Snippet: The lower wells had 600 μL lymphocyte media and 300 nM recombinant murine CXCL13 (R&D Systems).

Techniques: Cell Function Assay, Transplantation Assay, Incubation, Staining, Flow Cytometry, Labeling, Migration, Enzyme-linked Immunosorbent Assay, Positive Control, Two Tailed Test

Journal: BMC Immunology

Article Title: CXCL13 antibody for the treatment of autoimmune disorders

doi: 10.1186/s12865-015-0068-1

Figure Lengend Snippet: Effect on CXCL13- (A-D) and CXCL12- (negative control; E) induced migration of human and murine cells. Human pre-B-697-hCXCR5 cells (A, E) , human pre-B-697-hCXCR4 cells (E) , human tonsillar cells (B, E) , C57BL/6 splenocytes (C, E) , and SJL mouse splenocytes (D, E) were seeded into Transwell inserts. Diluted chemokines +/− antibodies were added into the lower chambers and plates were incubated for 2 h at 37°C (50 ug/ml of each antibody were used in CXCL12-induced migration assay). Inserts were removed and Alamar blue was added to the wells and incubated at 37°C overnight. Fluorescence was measured at wavelengths of 530 nm and 590 nm and the percent inhibition of chemokine-induced migration (A-D) or migration index (E) was calculated. Data represents an average of the measurements from at least three independent experiments + SEM. Statistical significance (relative to the isotype controls) was evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison post test **** P < 0.0001; *** P < 0.0003; **P < 0.01.

Article Snippet: Chemokines/Cytokines: recombinant human CXCL13 (Peprotech; 300–47); recombinant murine CXCL13 (Peprotech; 250–24); recombinant murine CXCL12 (Peprotech; 250-20A); recombinant human CXCL12 (Peprotech; 300-28A); recombinant murine IL-12 (R&D Systems; 419-ML/CF); recombinant murine IL-2 (R&D Systems; 402-ML); recombinant murine IL-6 (R&D Systems; 406-ML/CF); recombinant human TGF-β (R&D Systems; 240-B); recombinant murine IL-23 (R&D Systems; 1887-ML).

Techniques: Negative Control, Migration, Incubation, Fluorescence, Inhibition