Journal: Developmental cell

Article Title: Endocardially-derived macrophages are essential for valvular remodeling

doi: 10.1016/j.devcel.2019.01.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pentachrome staining was performed following the manufacturer’s protocols (Mastertech, KTRMP) and Hematoxylin and Eosin staining was performed following the standard protocol. table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti CD45 BD bioscience Cat#557659 Mouse monoclonal anti CD68 Bio rad Cat#852700 Mouse monoclonal anti F4/80 Bio rad Cat#MCA487A488 Mouse monoclonal anti CD206 Biolegend Cat#141716 Mouse monoclonal anti Csf1r Invitrogen Cat#14–1152-81 Mouse monoclonal anti aSMA Sigma Cat#A5228–200 Mouse monoclonal anti Vimentin Biolegend Cat#PCK-594P Mouse monoclonal anti CD45 BD biosciences Cat#557659 Mouse monoclonal anti CD11b eBioscience Cat#25011282 Mouse monoclonal anti F4/80 Biolegend Cat#123133 Mouse monoclonal anti CD64 BD Biosciences Cat#741024 Mouse monoclonal anti MHCII BD Biosciences Cat#563415 Mouse monoclonal anti CCR2 RnD Cat#FAB5538A Mouse monoclonal anti Ly6C BD Biosciences Cat#561237 Mouse monoclonal anti CX3CR1 Biolegend Cat#149007 Mouse monoclonal anti CD206 Biolegend Cat#141716 Mouse monoclonal anti CD117 eBioscience Cat#17–1171-83 Mouse monoclonal anti CD117 Biolegend Cat#105826 Mouse monoclonal anti Ly6G eBioscience Cat#15–5931-81 Mouse monoclonal anti CD41 BD Biosciences Cat#740504 Mouse monoclonal anti CD115 BD Biosciences Cat#743639 Mouse monoclonal anti CD31 BD Biosciences Cat#553372 Mouse monoclonal anti CD48 Biolegend Cat#103423 Mouse monoclonal anti Ly6A/Ly6E eBioscience Cat#45–5981-82 Mouse monoclonal anti CD150 Biolegend Cat#115929 Critical Commercial Assays DeadEnd TM Fluorometric TUNEL System Promega Cat#G3250 Phagocytosis Assay Kit (IgG FITC) Cayman chemical Cat#500290 Software and Algorithms FCS Express 4 De Novo software N/A JMP 11.0 SAS Institute N/A Excel 2016 Microsoft N/A PowerPoint 2016 Microsoft N/A Photoshop Adobe N/A Zen Zeiss N/A Open in a separate window KEY RESOURCES TABLE TUNEL apoptotic assay.

Techniques: TUNEL Assay, Phagocytosis Assay, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Haematopoietic reconstitution potential of spleen HSC for dendritic cells. Chimeras were prepared by adoptive transfer of marker-distinct neonatal spleen (SPL) cells along with host-type bone marrow (BM) cells into lethally irradiated mice. Whole splenocytes (1 × 10 5 ) from 8-day-old B6.SJL mice (CD45.1 + ) were co-injected into lethally irradiated (9.5Gy) C57BL/6J mice (CD45.2 + ) along with 1 × 10 5 host-type (CD45.2 + ) adult BM cells. Control BM chimeras were prepared by injection of only 1 × 10 5 dissociated CD45.1 + (B6.SJL) adult BM cells. For ‘short-term’ reconstitution, spleens were collected at 8 weeks, enriched for DC by depletion of T and B cells, and stained with antibodies specific for CD45.1, CD11c, CD11b, CD8 and MHC-II. Prior to flow cytometry, cells were incubated with propidium iodide (PI; 1 mg/ml) for gating of live (PI − ) cells. (A) Spleen APC subsets were gated as follows: L-DC (CD11c lo CD11b hi MHC-II − FSC hi ), myeloid cells (CD11c − CD11b hi MHC-II − FSC lo ), CD8a − cDC (CD11c hi CD11b + CD8 − MHC-II + ) and CD8a + cDC (CD11c hi CD11b − CD8 + MHC-II + ). (B) Spleen APC subsets were assessed for donor (CD45.1 + ) cell composition. Numbers in gates indicate per cent positive cells. C57BL/6J and B6.SJL splenocytes were included as negative and positive controls, respectively, in order to set gates to define CD45.1 − and CD45.1 + cell populations. Labelled chimeras (*) indicate mice showing only partial donor cell reconstitution.

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Adoptive Transfer Assay, Marker, Irradiation, Injection, Staining, Flow Cytometry, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Dendritic cell reconstitution by neonatal spleen HSC. Spleen (SPL) and bone marrow (BM) chimeras were prepared as described in . For analysis of long-term (.25 week) reconstitution, spleens were collected, enriched for DC by depletion of T and B cells, and stained with antibodies specific for CD45.1, CD11c, CD11b, CD8 and MHC-II. Prior to flow cytometry, cells were incubated with propidium iodide (PI; 1 mg/ml) for gating of live (PI − ) cells. C57BL/6J splenocytes were stained as a CD45.1 − control. (A) Individual spleen APC subsets (L-DC, myeloid cells, CD8 − cDC and CD8 + cDC) were gated as shown in and assessed for donor-cell (CD45.1 + ) composition. Numbers in gates indicate per cent positive cells. Labelled chimeras (*) indicate mice showing only partial donor reconstitution with some evidence of host-type cell development. (B) The relative prevalence of donor- to host-type cells was calculated for each spleen APC subset in animals showing partial donor reconstitution as indicated in and (*). The fold-increase of donor- to host-type cells was calculated relative to CD8 − cDC assigned a fold-increase of 1.0. Data represent mean 6 SE ( n = 4). Cell subsets having significantly higher representation of donor- over host-type cells, compared with CD8 − cDC, are indicated (*) ( P ≤ 0.02; Wilcoxon Rank Sum Test).

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Staining, Flow Cytometry, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Neonatal spleen HSC give multilineage long-term reconstitution. Mouse chimeras described in and were analysed for donor-derived (CD45.1 + ) subsets of L-DC, myeloid cells, CD8 − cDC, CD8 + cDC, CD8 + T cells, B cells, and ckit + haematopoietic progenitors in spleen, as well as CD8 + T cells and B cells in mesenteric lymph nodes (MLN). APC subsets were identified by antibody staining and flow cytometry as described in and . Lymphoid subsets in spleen were identified amongst the enriched subset discarded after T/B cell depletion to enrich for APC subsets. Cells were then stained for CD45.1, CD19 and CD8. Analysis of cells in MLN involved direct staining with antibodies specific for CD45.1, CD19 and CD8. For detection of haematopoietic progenitors, T/B cell depleted splenocytes were stained with antibodies specific for CD45.1, CD11c, CD11b, ckit and MHC-II. In all analyses, propidium iodide (PI; 1 mg/ml) staining was performed for gating of live (PI − ) cells. (A) The prevalence of donor-derived spleen L-DC, myeloid cells, CD8 − cDC and CD8 + cDC subsets in SPL and BM chimeras was calculated as a percentage of total (DC plus myeloid cells) in spleen. Chimeras were compared with control C57BL/6J mice for distribution of spleen APC subsets. (B) The presence of lymphoid cell subsets was analysed in spleen and MLN. For spleen, total [CD8 + T cells and CD19 + CD8 − B cells] are shown as per cent amongst T/B cell enriched spleen cells. For MLN, total [CD8 + T cells and CD19 + CD8 − B cells] are represented as per cent amongst total MLN. (C) Spleens collected from long-term SPL chimeras were analysed for the presence of Lin − ckit + progenitors. T/B cell depleted splenocytes (CD19 − Thy1.2 − ) were gated as CD11c − CD11b − MHC-II − (Lin − ) cells, prior to analysis of donor (CD45.1 + ) ckit expression. Numbers in gates indicate per cent positive cells.

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Derivative Assay, Staining, Flow Cytometry, Expressing