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Blood, Vol. 108, Issue 5, 1611-1617, September 1, 2006
Phosphatidylserine exposure in B lymphocytes: a role for lipid packing
Blood Elliott et al.
108: 1611
Supplemental materials for: Elliott et al
Files in this Data Supplement:
- Figure S1. Rates of cell shrinkage and decreased lipid packing are strain-dependent (PDF, 405 KB) -
i-viii) Lymphocytes from the strains indicated were labeled with anti-CD19APC or anti-CD19PERCP antibodies, as appropriate, to allow subsequent discrimination of B cells after mixing, mixed, and pre-incubated with 0.05 µM MC540 and stimulated with 0.2 µM calcimycin. Plots show number of MC540hi cells (left hand panels) and cell shrinkage (decrease in forward light scatter, FSC, right hand panels) plotted as a function of time. NOD and NZW lymphocytes, red lines; other lymphocytes, blue lines.
- Figure S2. Lymphocytes from CD45-/- mice show no evidence of increased incidence of apoptosis (PDF, 250 KB) -
Panel A. The level of active caspase 3 in B (CD19+) and T (CD4+) lymphocytes from parental (red line) and CD45ko (blue line) mice. Lymph node cells were labelled with anti-CD4 and anti-CD19 antibodies before fixing and permeabilisation (Pharmingen Cytofix/perm and Perm wash buffer) and stained for active caspase 3 (Pharmingen) (FL-1 units). As a positive control (green line), parental lymphocytes were treated with 50 µM etoposide for 4 hrs at 37°C before processing as above.
Panel B. Comparison of TdT-mediated dUTP nick end-labeling (TUNEL) staining of lymphocytes from parental (red line) and three different individual CD45ko mice (light blue, dark blue, orange lines). Cells were surface-labelled for CD19 and CD4 then analysed for TUNEL according to manufacturer’s protocol (Roche). As a positive control, parental lymphocytes were treated with (2000 U/ml) DNase I and processed as above. Incorporation of dUTPFITC was indicated by increased FL-1 fluorescence and was assessed by flow cytometry.
Panel C. Comparison of mitochondrial transmembrane potential (MTP) in lymphocytes from parental (red line), and several individual CD45ko mice (CD19+; light blue, dark blue and yellow lines: CD4+ dark blue line) as measured by DiOC6 accumulation. Splenocytes were surface stained with anti-CD19CYCHROME and anti-CD4APC antibodies then loaded with DiOC6 (25ng/ml) for 15 min at 37°C 5% CO2 in air. Cells were washed and analysed immediately. As a positive control, DiOC6 accumulation was compared with that by splenocytes (not stained with anti-CD4 and anti-CD19) treated with 50 µM etoposide for 4 hrs at 37°C (green line).
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