Standardization of the 2-color flow-FISH technique.

The effect of heating on FSC and SSC profiles was investigated. Control PBMCs were fixed with 2% paraformaldehyde and analyzed before (A) and after (B) heating at 80°C for 10 minutes. Gates for telomere analysis were determined by the addition of propidium iodide for the exclusion of cell debris from dead/dying cells, and also doublets and blastoid cells (data not shown). Cells within the R1 gate were stained with anti-CD8 coupled to Cy5, fixed, and analyzed before and after heating to 80°C (C). Although the MFI for CD8-Cy5 decreased from 167 for the control to 78 in the heated sample, percentage of CD8⁺ T cells was relatively unaffected by the heating protocol (23.6% before, 19% after). PBMCs were stained with anti-CD8 biotin followed by streptavidin-Cy5. Cells were then fixed and permeabilized and processed for flow-FISH as described in “Patients, materials, and methods” with either the control X-chromosome FITC PNA probe (D) or the FITC-labeled PNA telomeric probe (E).