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: Frontiers in Immunology

Article Title: Improved Functionality of Exhausted Intrahepatic CXCR5+ CD8+ T Cells Contributes to Chronic Antigen Clearance Upon Immunomodulation

doi: 10.3389/fimmu.2020.592328

Figure Lengend Snippet: Exhausted Ova-specific CD8+ T cells in the liver harbor a CXCR5+ CD8+ T cell subset. (A) Representative gating strategy employed to identify Ova-specific CXCR5+ T cells using fluorescence minus one (FMO) control. (B, C) Frequencies (B) and absolute numbers (C) of CXCR5+ Ova-specific CD8+ T cells in the liver of high antigen mice post AdOva vaccination (see Figure S1B for experimental details). At indicated time points after vaccination, liver non-parenchyma cells were isolated, and phenotyped for CXCR5+ Ova specific CD8+ T cells. Stained cells were analyzed by flow cytometry. (D) Frequency of Bcl6 expression in naïve, Ova- specific CXCR5+ and CXCR5- CD8+ T cells 21 days post vaccination. (E) MFI of Bcl6 expression in naïve, Ova-specific CXCR5+ and CXCR5- T cells in (C) . Representative histogram overlay (left) and summary of Bcl6 MFI (right). (F) Serum concentration of CXCL13 from AdOva vaccinated wildtype and high antigen in mice, as well as high antigen mice without vaccination. (G) Frequency of LFA-1 (CD11b) on CXCR5+ and CXCR5- CD8+ T cells 21 days post OT-1 transfer. (H) MFI of LFA-1 expression in (G) . Representative histogram plot (left) and summary (right) of LFA-1. (I) Frequency of Nur77 expression in CXCR5+ and CXCR5- Ova-specific CD8+ T cells. (J) MFI of Nur77 expression in naïve, CXCR5+ and CXCR5- CD8+ T cells. Representative histogram plot (left) and summary (right) of Nur77. *p ≤ 0.05; **p ≤ 0.01; ns: p > 0.05.

Article Snippet: CXCL13 was quantified using the murine BLC (CXCL13) ELISA kit (Thermo Scientific) according to the manufacturer’s protocol.

Techniques: Fluorescence, Control, Isolation, Staining, Flow Cytometry, Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Levels of Murine, but Not Human, CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7

doi: 10.1371/journal.pone.0072414

Figure Lengend Snippet: Immunohistochemistry was performed as described in the Materials and Methods , using an HRP/DAB color development system with hematoxylin counterstaining. Some sections were stained with both hematoxylin and eosin (H and E). Typical staining results for each marker are given in the figure as follows: ( A ) H and E, ( B ) human CXCR5, ( C ) a representative negative control, ( D ) human CXCL13, ( E ) murine CXCL13, and ( F ) F4/80. The results of the H&E staining in ( A ) show a tumor that in many respects resembles human BL, which typically contain significant numbers of infiltrating macrophages, giving a “starry sky” appearance (see arrows). The corner insert in ( A ) shows a 3-fold enlargement of the area in the image to which the upper arrow points, with such a macrophage in the center of the enlargement. The image in ( E ) shows that tumors appear to be infiltrated with scattered cells that stain positive for murine CXCL13. In an attempt to more precisely determine the cell type of these infiltrating cells serial tumor sections were stained separately for murine CXCL13 and for F4/80, and images of the resulting staining were enlarged 3-fold compared to the images in ( A – F ) to provide more detail ( G , H ). In ( G ) and ( H ), both mCXCL13(+) and F4/80(+) cells appear to be large cells that sometimes have dendritic processes. All images shown in this figure were taken at an original magnification of 200X. Each image contains an internal scale marker that is 100 microns in length.

Article Snippet: Levels of murine and human CXCL13 in serum and in tumor ascites fluid were determined by ELISA, using kits validated to be specific for either murine or human CXCL13 (QuantikineTM, R&D Systems), with no cross-reactivity.

Techniques: Immunohistochemistry, Staining, Marker, Negative Control

Journal: PLoS ONE

Article Title: Levels of Murine, but Not Human, CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7

doi: 10.1371/journal.pone.0072414

Figure Lengend Snippet: ( A ) Murine CXCL13 levels in the serum of control mice (n = 13) and in the serum of mice with tumors (n = 14), as determined by ELISA. ( B ) Murine CXCL13 levels in the ascites fluid of the mice with tumors. The solid square within each box plot represents the mean value for the data. In ( A ), there is an ~20-fold increase in mean mCXCL13 levels in the serum of mice with tumors compared to control mice (p < 0.0001; t-test). In ( B ), the long lower whisker in the box plot of the data reflects the presence of several low outliers; these lower values were generally seen in animals that developed smaller, more localized tumors at the site of injection as opposed to widespread metastatic disease.

Article Snippet: Levels of murine and human CXCL13 in serum and in tumor ascites fluid were determined by ELISA, using kits validated to be specific for either murine or human CXCL13 (QuantikineTM, R&D Systems), with no cross-reactivity.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Whisker Assay, Injection