At the stage of transplantation 99.92% of all cells were viable, as revealed by Via Probe staining.
We analyzed the tetramer expression within the CD4+T cell subset by gating on the lymphocytes and excluding B cells, monocytes and dead cells (via probe positive population).
Additionally, to generate circular polarization (CP), the patch has been reduced diagonally and shorted by a via-probe.
Cell viability was confirmed to be acceptable using a 7-aminoactinomycin D dye exclusion assay (BD Via-Probe, BD Biosciences, San Jose, CA, USA) and flow cytometry (Accuri C6, BD Biosciences, San Jose, CA, USA).
Cell death was confirmed by flow cytometry using Annexin V and Via-Probe staining (BD Biosciences).
Under these conditions of hypoxia we did not observe apoptotic or necrotic H9C2 cells as determined by staining for annexin V and Via-probe (not shown).
Live, late-apoptotic and necrotic cells (as determined by staining with annexin V and Via-probe) were incubated with increasing concentrations of purified C4BP-PS and the binding was analyzed by flow cytometry.
Apoptosis of the trastuzumab treated versus non treated cells was measured by using the following antibodies: anti-human Annexin-V APC conjugated (ICQ, Groningen, The Netherlands), in order to quantify apoptosis; Via-probe 7aad (necrosis marker; R&D System), in order to exclude necrotic cells from the final analysis.
The cell states were confirmed with Annexin V (Immunotools, Friesoythe, Germany) and Via-Probe (BD, San Jose, CA, USA) staining.
To analyze DNA degradation, we used Hoechst (Invitrogen, Waltham, MA, USA) or Via-Probe to detect cellular double stranded DNA (dsDNA).
