Facsdiva software 6

Externalization of PS in the treated cancer cells was evaluated by staining with Alexa Fluor 488—annexin V (Apoptosis kit, Invitrogen, USA) in accordance with the manufacturer’s instructions. In addition to fluorescence microscopy, the intensity of cellular fluorescence was also measured by flow cytometry on a BD FACS ARIA II machine with BD FACS Diva software 6.1.3.

Cell characteristics and culture conditions of FH-hTERT were described previously [18 (link)]. Generation of lentiviral vectors, vector plasmids and lentiviral-vector mediated RGB marking of FH-hTERT by LeGO-C2 (Addgene No. 27339), LeGO-V2 (Addgene No. 27340) and LeGO-Cer2 (Addgene No. 27338) were described in detail [14 (link), 15 (link)]. 5×10⁴ or 1×10⁶ cells were transduced at a multiplicity of infection (MOI) of 1 for each vector. In order to eliminate untransduced FH-hTERT, fluorescence-positive cells were sorted by fluorescence-activated cell sorting (FACS) on a FACS AriaIIIu (BD Biosciences, Heidelberg, Germany), using three different BP Filters (670 LP, 530/30, 510/50 nm). Flow-cytometry analysis was performed using FACS Canto II and BD FACS Diva Software 6.1.3 (both BD Biosciences).

The genome size of cell populations from the testes was estimated by measurement of the cell nuclei using a BD FACSAria II flow cytometer on a subset of 17 individuals (same individuals as used for karyotype analyses; table 1 and supplementary table S1, Supplementary Material online). Testes and muscle tissues were fixed in 70% ethanol prior to measurements. Testes and muscle tissues were minced in 0.1% Triton X100, 10 µ/ml DAPI and 15 mM MgCl₂ to release nuclei from cells. These nuclei suspensions were incubated at +4 °C overnight. After incubation of nuclei suspensions for 4–6 h (at +4 °C), they were analyzed by BD FACSAria flow cytometer. At least 10,000 events were measured. BD FACSDiva software (6.1.3) was used to process the obtained data. Suspension of nuclei released from muscle cells was used as an internal control.

Mononuclear cells were immunostained with various combinations of fluorescent antibodies against CD3, CD4, CD8, IL-17, Foxp3, Ki-67, CD11b, CD8α, and granzyme B (eBioscience, San Diego, CA, United States). For intracellular cytokine detection, the cells were restimulated with PMA and ionomycin for 4 h in the presence of Golgi-stop. Labeled cells were enumerated by Canto II flow cytometer (BD Biosciences, San Jose, CA, United States). Data were analyzed by BD FACSDiva software 6.0 (BD).

Apoptosis was evaluated by 7-AAD staining, followed by flow cytometry analysis. HUVE cells were plated at a density of 2.5 × 10⁵ cells/well on 6-well plates and grown overnight. The following day, the cells were treated either with XN or EGCG in a concentration range from 5 to 20 μM, up to 96 hours. DMSO in medium or medium alone were used as vehicle. At each time point, cells were harvested, counted, transferred into flow tubes, pelleted, resuspended in 50 μl of PBS and stained with the 7-aminoactinomycin-D (7-AAD) dye. Samples were analyzed by flow cytometry within 1 hour using a FACSCantoII (BD). The proportion of viable (7-AAD-negative) cells was analyzed using the BD FACSDiva Software 6.0 and expressed as a percentage of the total cell number, excluding debris.

S. mutans that has been exposed to the compounds or control bacteria were washed in PBS and incubated with various fluorescent dyes according to the aim of the experiment before being analyzed on flow cytometer. A 20 min staining solution of 3.3 µM SYTO 9 together with 2 µg/mL PI was used to analyze bacteria with disrupted membrane [35 (link)]. This staining was performed at room temperature. For determining the membrane potential, the bacteria were exposed to 15 μM 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)) solution in PBS for 30 min at room temperature prior to flow cytometry [20 (link)]. For determining the membrane and DNA content, the bacteria were exposed to 10 µg/mL Nile red (APExBIO, Houston, TX, USA) and 1 µg/mL DAPI (Sigma) for 30 min at 37 °C [20 (link)]. The following excitation/emission parameters were used: 488 nm/531 nm for SYTO 9 and the green fluorescence of DiOC2(3); 488 nm/620 nm for PI and the red fluorescence of DiOC2(3); 561 nm/635 nm for Nile red and 355 nm/450 nm for DAPI. The samples were analyzed by the LSR-Fortessa flow cytometry instrument (BD Biosciences, San Jose, CA, USA), and 50,000 events were collected using the BD FACSDiva software 6.0. The FCS Express 7 software was used for analyzing the data.