fcblock clone 2.4g2  (Bio X Cell)

Journal: European Journal of Immunology

Article Title: Depletion of CD11c + cells in the CD11c.DTR model drives expansion of unique CD64 + Ly6C + monocytes that are poised to release TNF-α

doi: 10.1002/eji.201545789

Figure Lengend Snippet: Expansion of Ly6C + monocytes following depletion of CD11c + cells. CD11c.DTR mice were injected with PBS or DT and analyzed 48 h later. (A) Representative dot plots showing the expansion of monocytes (blue and orange) and neutrophils (green) in the spleens of PBS- and DT-injected CD11c.DTR mice. Dot plots are pregated on live single cells. Dot plots on the far right show expression of Ly6C and Ly6G by CD11b + Ly6C high (blue and orange) and CD11b + Ly6C int (green) gated cells overlaid on live cells. (B) Representative dot plots showing expression of CD115 and Ly6C by gated CD11b + Ly6C high (blue and orange) or Ly6C int (green) cells. Plots are representative of more than three independent experiments. (C) Graphs show the percentage ± SEM (left) and number ± SEM (right) of CD11b + Ly6C high monocytes in the spleen of PBS-treated (blue) and DTR-treated (orange) recipients at different times post injection of DT. Two-way ANOVA with multiple comparisons (*) PBS versus DT on day 2 p < 0.0001. Data are pooled from multiple independent experiments: day 1 PBS/DT n = 6, day 2 PBS/DT n = 26/24, day 4 PBS/DT n = 6. (D) Left: graph showing the frequency ± SEM of monocytes in the blood and BM of PBS-treated (blue triangles and squares) and DT-treated (orange triangles and squares) recipients. Two-way ANOVA with multiple comparisons (*) day 2 BM and blood p = 0.0016 and p = 0.0012, respectively, day 4 p < 0.0001 for both. Right: graph showing the number ± SEM of monocytes in the BM from the femurs and tibias of one back leg. Two-way ANOVA with multiple comparisons (*) p = 0.0015 on day 4. Data are pooled from multiple independent experiments: days 1 and 4 PBS/DT n = 6, day 2 BM PBS/DT n = 14/12, blood PBS/DT n = 12.

Article Snippet: The following antibodies were used for flow cytometric analysis, all from eBiosciences or BD-Biosciences: FcBlock (clone 2.4G2), anti-CD11b (M1/70), anti-CD11c (HL3), anti-Ly-6C (AL-21), anti-Ly-6G (1A8), anti-CD45.1 (A20), anti-CD45.2 (104), anti-MHCII (M5/114.15.2), anti-CD115 (AFS98), anti-CD64, anti-F4/80 (BM8), anti-CD135 (A2F10), anti-CD117 (2B8).

Techniques: Injection, Expressing

Journal: European Journal of Immunology

Article Title: Depletion of CD11c + cells in the CD11c.DTR model drives expansion of unique CD64 + Ly6C + monocytes that are poised to release TNF-α

doi: 10.1002/eji.201545789

Figure Lengend Snippet: DT-Ly6C + monocytes express CD64. (A) Photomicrographs show Giemsa-stained cytospins of sorted CD11b + Ly6C high CD115 high cells from PBS- and DT-treated mice, bars = 100 μM. (B) Representative histograms (top) and graph (bottom) showing SSC of CD11b + Ly6C high CD115 high monocytes from PBS-treated (white) or DT-treated (gray) mice. Graph shows SSC ± SEM from PBS-treated ( n = 15) and DT-treated ( n = 13) mice. Data are pooled from four independent experiments: PBS versus DT p = 0.0186. (C) Top: representative histograms showing expression of labeled surface markers on CD11b + Ly6C high or CD11b + Ly6C high CD115 high monocytes from PBS-treated (white) and DT-treated (gray) mice. Staining with the relevant isotype control is shown by the thin line. Numbers are the median fluorescence intensity (MFI). Bottom: Graphs show the MFI ± SEM for CD64, CD11c, MHC II, and F4/80 on monocytes gated as for the histograms ( n = 8 or 6 for F4/80 expression). Horizontal lines mark the mean; data are pooled from three independent experiments: CD64 PBS versus DT p = 0.0002, CD11c p = 0.0014, MHC II p = 0.0070, F4/80 p = 0.0043. (D) Left: representative dot plots showing the frequency of gated live cells in the blood of CD11c.DTR mice injected with DT 48 h earlier. Right: histograms showing expression of CD64 by CD11b + Ly6C high monocytes from PBS-treated (white) and DT-treated (gray) mice. (E) Paired line graphs showing the median fluorescent intensity of CD64 on CD11b + Ly6C high monocytes from the blood and spleens of PBS- and DT-treated mice. Data are pooled from three independent experiments, n = 8. Student's paired t -test in both PBS- and DT-treated animals, p < 0.0001. Statistical analyses for other experiments were carried out with a Student's unpaired t -test (+) or Mann–Whitney (*) as appropriate.

Article Snippet: The following antibodies were used for flow cytometric analysis, all from eBiosciences or BD-Biosciences: FcBlock (clone 2.4G2), anti-CD11b (M1/70), anti-CD11c (HL3), anti-Ly-6C (AL-21), anti-Ly-6G (1A8), anti-CD45.1 (A20), anti-CD45.2 (104), anti-MHCII (M5/114.15.2), anti-CD115 (AFS98), anti-CD64, anti-F4/80 (BM8), anti-CD135 (A2F10), anti-CD117 (2B8).

Techniques: Staining, Expressing, Labeling, Fluorescence, Injection, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: Depletion of CD11c + cells in the CD11c.DTR model drives expansion of unique CD64 + Ly6C + monocytes that are poised to release TNF-α

doi: 10.1002/eji.201545789

Figure Lengend Snippet: DT-Ly6C + monocytes are transcriptionally distinct from blood and tissue monocyte populations. CD11c.DTR mice were injected with PBS or DT, and 48 h later splenic lineage neg CD11b + CD115 high Ly6C high monocytes were sorted to high purity and analyzed by hybridization to an Affymetrix microarray. We also sorted lineage neg CD11b + CD115 high Ly6C neg monocytes and BM cMoPs (lineage neg CD117 + CD115 + CD135 neg Ly6C + CD11b neg ) from C57BL/6 mice. (A) Multidimensional Scaling (MDS) comparing the transcriptional profile of DT-Ly6C + cells to other precursors and monocyte populations (Immgen MDP n = 3, UCL cMoP n = 3, UCL PBS Ly6C + n = 4, and Immgen (PBS) Ly6C + n = 3, UCL Ly6C neg n = 3, and Immgen n = 2). Each subset of cells was purified from at least two independent experiments and was derived from individual mice, or pooled from multiple mice. (B) Heat map comparing the relative expression of a panel of genes associated with monocyte differentiation in the different cell populations. (C) PCA of gene expression by DT- and PBS-Ly6C + monocytes in comparison to published data for CD64 + migrating LN monocyte-derived DCs and dermal-cell subsets . Approximately, 45 and 13% of the variance within the expression data is orthogonal to PC1 and PC2, respectively (58% cumulative). (D) Heat map depicting the relative expression across all samples of the principal gene signature (PC1, Supporting Information Fig. ) that separates the dermal P1–P5 group. DT-Ly6C + n = 4, PBS-Ly6C + n = 9 (Immgen, UCL), P1–P5 .

Article Snippet: The following antibodies were used for flow cytometric analysis, all from eBiosciences or BD-Biosciences: FcBlock (clone 2.4G2), anti-CD11b (M1/70), anti-CD11c (HL3), anti-Ly-6C (AL-21), anti-Ly-6G (1A8), anti-CD45.1 (A20), anti-CD45.2 (104), anti-MHCII (M5/114.15.2), anti-CD115 (AFS98), anti-CD64, anti-F4/80 (BM8), anti-CD135 (A2F10), anti-CD117 (2B8).

Techniques: Injection, Hybridization, Microarray, Purification, Derivative Assay, Expressing

Journal: European Journal of Immunology

Article Title: Depletion of CD11c + cells in the CD11c.DTR model drives expansion of unique CD64 + Ly6C + monocytes that are poised to release TNF-α

doi: 10.1002/eji.201545789

Figure Lengend Snippet: DT-Ly6C + monocytes express genes associated with innate immune activation, and secrete high levels of TNF-α upon stimulation with LPS. (A) Gene set enrichment analysis was used to identify differentially expressed pathways between DT-Ly6C + cells and their most closely related group: PBS-Ly6C + . The rank-versus-ES score plots are shown for two representative pathways upregulated in DT-Ly6C + cells: blue = Reactome toll receptor cascade, red = Reactome innate immune system. All pathways were significantly altered (false discovery rate q -values < 0.01). (B) Table showing the core enriched genes for the two pathways. (C) Purified CD11b + Ly6C + CD115 high monocytes from PBS-treated ( n = 6) or DT-treated ( n = 6) treated mice were cultured overnight in medium with or without LPS. Graphs show the amount ± SEM of cytokines secreted into the culture supernatant. TNF-α PBS versus DT for LPS-treated cells, p = 0.022. Data are pooled from two independent experiments. Statistical analyses were carried out with a Student's unpaired t -test.

Article Snippet: The following antibodies were used for flow cytometric analysis, all from eBiosciences or BD-Biosciences: FcBlock (clone 2.4G2), anti-CD11b (M1/70), anti-CD11c (HL3), anti-Ly-6C (AL-21), anti-Ly-6G (1A8), anti-CD45.1 (A20), anti-CD45.2 (104), anti-MHCII (M5/114.15.2), anti-CD115 (AFS98), anti-CD64, anti-F4/80 (BM8), anti-CD135 (A2F10), anti-CD117 (2B8).

Techniques: Activation Assay, Purification, Cell Culture