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Blood, Vol. 109, Issue 10, 4220-4228, May 15, 2007
TC-PTP–deficient bone marrow stromal cells fail to support normal B lymphopoiesis due to abnormal secretion of interferon-
Blood Bourdeau et al.
109: 4220
Supplemental materials for: Bourdeau et al, Vol 109, Issue 10, 4220-4228
Files in this Data Supplement:
- Figure S1. Phenotypic analysis of cultured bone marrow stromal cells (PDF, 52.1 KB) -
Flow cytometry analysis of bone marrow stromal cell cultures, passage 5, obtained from TCPTP+/+ and TC-PTP−/− mice at P7. The relative cell number (RCN) is plotted against the fluorescence of each marker indicated. Representative histograms are shown.
- Figure S2. Analysis of bone marrow immature and mature recirculating B cells (PDF, 44.6 KB) -
Whole bone marrow was harvested from TC-PTP+/+ and TC-PTP−/− mice at P7 (4 WT, 3 KO). (A) Cells were stained for surface expression of CD25, CD43, B220, IgM, and IgD and analyzed by flow cytometry. Analysis was gated on B220+, CD25−, and CD43− cells to include only the pooled population of immature/mature recirculating B cells. The discrimination between these B-cell subpopulations was achieved with IgM and IgD. The percentages of immature and recirculating mature B cells are provided for TC-PTP+/+ and TC-PTP−/− mice. Representative data are shown. (B) Total absolute B-cell counts and for each B-cell subset from TC-PTP+/+ (□) and TCPTP−/− mice (■) are illustrated. Cell counts were derived by multiplying the percentage of total cells, obtained by flow cytometry for each subpopulation, by the whole marrow cell counts, obtained by manual counting with hemocytometer. Cell counts are reported as mean ± SD. *P < .01. All experiments were repeated at least 3 times.
- Figure S3. Characterization of in vitro pre-B–cell cultures (PDF, 71.4 KB) -
Whole bone marrow was harvested from TC-PTP+/+ and TC-PTP−/− mice at P7 (3 WT, 3 KO). Pre-B cells were obtained from a 5-day bone marrow culture in the presence of IL-7. (A) Absolute cell counts were determined from TC-PTP+/+ (□) and TC-PTP−/− (■) culture by manual counting of nonadherent cells with a hemocytometer. Cell counts are reported as mean ± SD. *P < .01. (B) Pre-B cells were stained for surface expression of CD25, CD43, B220, and with annexin V and propidium iodide (PI) and analyzed by flow cytometry. The number of cells is plotted against the fluorescence of B220 to assess the purity of the cultures, whereas the analysis of B-cell subsets by CD43/CD25 and apoptosis by annexin V/PI were gated on B220+ cells. The percentages of large pre-B cells (CD43+, CD25+), small pre-B cells (CD43−, CD25+), preapoptotic cells (annexin V+, PI−), and apoptotic cells (annexin V+, PI+) are provided. All experiments were repeated at least 3 times. (C) Day 5 bone marrow pre-B–cell cultures were labeled with CFDA-SE for 5 days and stained for surface expression of B220 for flow cytometry. Analysis was gated on B220+ cells to include only B cells. The RCN is plotted against CFDA-SE fluorescence. The number of cell divisions is indicated next to the corresponding CFDA-SE peak.
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