Fcs express v3

Flow cytometry analysis was performed using a BD LSRII flow cytometer. To detect live HeLa cells transfected with the pGAF-FP-sfGFP-N1 plasmid, a 488 nm Ar gas laser line, a 640 nm solid-state laser, and the 530/40 nm and 670 nm long pass emission filters were used. To quantify the NIR fluorescence brightness of the transiently transfected cells, the mean NIR fluorescence signal was normalized to the absorbance spectra and the extinction coefficients of GAF-FP and to the transmission of the 670 nm long pass emission filter. The histograms of cell populations and mean NIR fluorescence intensities of the analyzed cells were obtained using the FCS express v.3 software (DeNovo Software).

Cell apoptosis assay was performed using KeyGEN Annexin V-APC/propidium iodide (PI) Apoptosis Detection kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer's instructions. Briefly, cells were treated with 5 µM or 10 µM imatinib for the indicated times. The samples were washed with ice cold phosphate-buffered saline (PBS) and stained with 2.5 µg/ml Annexin V-APC and 1 µg/ml propidium iodide. The samples were then examined by fluorescence-activated cell sorter (BD Bioscience, San Jose, CA, USA) as previously described [5] (link), [29] . For cell viability assay, cells were treated with 10 µM imatinib for indicated time, washed with ice cold PBS, stained with 1 µg/ml of propidium iodide, and examined by fluorescence-activated cell sorter as described previously [5] (link), [29] . All the data were analyzed by FCS Express V3 Software (De Novo Software, Thornhill, Ontario, Canada).

Apoptosis assay was performed as previously described [4 (link), 5 (link)]. Briefly, cells were cultured with certain inhibitors for indicated time. Then cells were stained with 2.5 μg/ml Annexin V-FITC and 1 μg/ml PI. For cells overexpressing or silencing given genes, samples were stained with 2.5 μg/ml Annexin V-APC and 1 μg/ml 7-AAD. Samples were examined by fluorescence-activated cell sorter (FACS) (BD Bioscience) as previously described [5 (link)] and data were analyzed by FCS Express V3 Software (De Novo Software). For experiments using combined treatment, the synergism was evaluated by the combination index (CI) which was calculated using Chou-Talalay method by CompuSyn software (ComboSyn, Inc.) [49 (link), 50 (link)]. Briefly, CI value < 1 represents synergistic effect, CI values = 1 additive effect, and CIs > 1 antagonistic effect.

After cell treatment, the cells were detached from the plates with trypsin and resuspended for live cell analysis or fixed for 5 min with cold (−20 °C) methanol. Fixed cells were washed with DPBS (Lonza) before resuspending in DPBS for analysis. About 10,000 cells were analyzed for each condition. The flow cytometry experiments were conducted with a BD Fortessa Cell Analyzer (BD Biosciences) and data were analyzed using the FCS Express v3 software (De Novo Software). The Students’ t-test was used to compare the mean fluorescence values of different conditions and in addition the effect size was calculated using formula d = (M₁-M₂)/SD where d is Cohen’s effect size index, M₁-M₂ is the difference between the group means and SD is the standard deviation of either group. The effect sizes were classified to very small (<0.2), small (0.2–0.4), medium (0.5–0.7), large (0.8–1.2) and very large (>1.3)^{64 (link)}.

After 384 days of incubation, subsamples (1 mL) were taken from each dialysis bag to determine viral and host abundances. They were fixed with glutaraldehyde (0.5% final concentration) and incubated at 4°C for 15 min in the dark. After flash-freezing in liquid nitrogen, the samples were stored at –80°C before analysis. By analyzing flow cytometry (Epics Altra II, Beckman Coulter) scatter plots for side scatter versus red fluorescence and for orange fluorescence versus red fluorescence, the Prochlorococcus and Synechococcus abundances could be directly determined (83 (link)). After cells were stained with 1.0 × 10^–4 SYBR Green I (vol/vol, final concentration of the stock solution, Molecular Probes) and then incubated for 15 min in the dark, heterotrophic bacterial abundance was determined by flow cytometry with scatter diagrams of side scatter versus green fluorescence (83 (link)). In addition, subsamples were diluted with Tris-EDTA buffer (pH 8.0; Sigma, St. Louis, MO, USA), stained with 5.0 × 10^–5 SYBR Green I (vol/vol, final concentration of the stock solution), and incubated at 80°C for 10 min. Then viral abundance was determined by flow cytometry with a scatter diagram of side scatter versus green fluorescence (81 (link), 82 (link)). Finally, all data analysis was performed with FCS Express V3 software (De Novo Software, Los Angeles, CA, USA).

Lysosomal damage by DSS challenge was evaluated by acridine orange stain as described elsewhere [17 (link)]. Briefly, macrophages were plated into 24-well culture dishes overnight and introduced to apoptotic cells (1:8) for 2 h. Macrophages were then washed, primed with LPS, and stimulated for 24 h with DSS. Cells were then washed and incubated with 0.25μg/mL acridine orange for 15 min for lysosome stain. Lysosomal damage was determined as loss of fluorescence intensity emission at 600–650 nm with an LSRII (BD Biosciences). Data analysis was performed using FCS express v3 software (De Novo Software).