Hoechst 34580

Cytotoxicity and apoptosis of reprogrammed and naive CD8⁺ T-lymphocytes were studied in cell culture of CSCs isolated from adhesive fraction of mononuclear cells from patient with SCLC. After co-incubation, reprogrammed and naive human CD8⁺ T-lymphocytes and CSCs were stained with Hoechst 34580 (for nuclear staining, Becton Dickinson, San Jose, CA, USA) and 7-AAD (for apoptotic cells detection, Becton Dickinson, San Jose, CA, USA). Preliminary reprogrammed and naive human CD8⁺ T-lymphocytes were stained with CFSE during 24 h. CFSE staining of CD8⁺ T-lymphocytes was performed according to the manufacturer’s instruction (Becton Dickinson, San Jose, CA, USA). Cytotoxicity of CD8⁺ T-lymphocytes in CSCs culture was assessed by analysing the ratio of cells counted in the blue and yellow channels to the total number of CSCs (percentage of dead CSC Hoechst⁺7-AAD⁺). Determination of the percentage of dead CD8⁺ T-lymphocytes Hoechst⁺CFSE⁺7-AAD⁺ was made by the ratio of cells counted in blue and green channel to total cells.
All images were obtained with Cytation 5 (4× or 20× magnification) followed by cell analysis using Gen5™ data-analysis software (BioTek, Instruments, Friedrichshall, Germany). Prior to the analysis, images were pre-processed to align the background.

For confirmation of the presence of CSC markers on single cells and tumor spheroids formed during the cultivation of the adhesive fraction of mononuclear cells obtained from a patient with SCLC, staining with fluorophore-conjugated monoclonal antibodies was performed (CD87 BV421, CD117 BB515, CD274 BV421, Axl BV480, EGF Receptor Alexa Fluor^® 647, and CFSE, all Becton Dickinson, San Jose, CA, USA)). Spheroids and single cells were stained with Hoechst 34580 (for nuclear staining, Becton Dickinson, San Jose, CA, USA), CD87, CD117, CD274, Axl, EGF, and/or CFSE during 30 min. Dyes were used in various combinations (Hoechst/CFSE/EGF) (CD87/CD117/EGF), (Axl/CD117/EGF), (CD274/CD117/EGF). CSCs culture was assessed by analyzing the ratio of cells counted in the blue and/or green/yellow channels. Tumor spheroids were defined as a three-dimensional cellular structure based on images obtained using Cytation 5 (4× or 20× magnification) followed by cell analysis using Gen5™ data analysis software (BioTek, Instruments, Friedrichshall, Germany). Prior to the analysis, images were pre-processed to align the background.
Before staining analysis of single cells, tumor spheroids were excluded by size using the Gen5™ data-analysis software interface (BioTek, Instruments, Friedrichshall, Germany).

Cells collected from the spleen, bone marrow, or blood were washed twice with PBS containing 1% BSA and stained with CD45, TER-119, and CD71 fluorescent antibodies. Then, cells were incubated with Hoechst 34580 (0.5 µg/ml, BD) at room temperature in dark for 30 min to be measured by flow cytometry. In CD45^- Ter119⁺ cells, CD71^- indicated mature and enucleated erythroid cells, and CD71⁺ indicated less mature erythroid progenitor cells that may contain DNA molecules. Hoechst, a DNA dye with cell- permeability, could stain parasite and erythroblast DNA. In CD45^- Ter119⁺ CD71^- population, uninfected mature erythroid cells without DNA molecules are Hoechst^neg (CD45^- Ter119⁺ CD71^- Hoechst^neg), infected mature erythroid cells that only contain parasite DNA are Hoechst^pos (CD45^- Ter119⁺ CD71^- Hoechst^pos). In CD45^- Ter119⁺ CD71⁺ population, uninfected and relatively mature erythroid progenitor cells without DNA molecules are Hoechst^neg (CD45^- Ter119⁺ CD71⁺ Hoechst^neg), uninfected and less mature erythroid progenitor cells that only contain erythroblast DNA are Hoechst^low (CD45^- Ter119⁺ CD71⁺ Hoechst^low), infected and less mature erythroid progenitor cells that contain erythroblast and parasite DNA are Hoechst^hi (CD45^- Ter119⁺ CD71⁺ Hoechst^hi).