Figure 1

Telomere length (TL) measurement using flowFISH on proliferating T lymphocytes depends on fixation-permeabilization and RNA nuclease treatment. PBMC from healthy adults were either stimulated with immobilized anti-CD3 and anti-CD28 for 4 days or were cryopreserved on day 0 and thawed for processing on day 4; CD3⁺ T cells were magnetically sorted from both samples on day 4. Each sample was divided for flowFISH TL analysis and telomere restriction fragment (TRF) Southern blotting. (A) FlowFISH analysis using 3 different pre-hybridization conditions: without fixation-permeabilization prior to probe hybridization (no fix/perm), with fixation-permeabilization (Fix/Perm) prior to hybridization, and with fixation-permeabilization followed by RNase treatment prior to hybridization (Fix/Perm +RNase). Statistical comparisons were done using unpaired t-test, *** p<0.001, and ns=not significantly different. (B) TRF Southern blot analysis. DNA was extracted from CD3+ T cells isolated ex vivo or after stimulation with anti CD3+CD28 for four days and subject to Southern Blot Analysis. TRF lengths are shown at the bottom of both lanes and are the average of three separate 20 pixel-wide analyses using MatLab software running the MaTelo macro (see Methods).