Facsverse cytometer

Halothece sp. cells at the end of the different experiments were fixed with glutaraldehyde 25% (v/v) in H₂O (Sigma-Aldrich) [final concentration 0.05% (v/v)] and were counted in a Becton Dickinson FACSVerse cytometer (Becton and Dickinson, Franklin Lakes, NJ, United States). Fluorescent beads, BD FACSuite^TM CS&T research beads (Becton and Dickinson and Company BD Biosciences, San Jose, CA, United States), were used as internal standard to calibrate the instrument. To count Halothece sp. cells, we selected fluorescein isothiocyanate (FITC) (488-nm excitation, 530/30-nm emission) and phycoerythrin (PE) (488-nm excitation, 576/26-nm emission) combination fluorescence signals, which show clearly the population of the cells, recording for each sample a total of 10 × 10³ cells.

A preliminary study of viability was conducted using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. For this assay, 5 mg of MTT (Sigma-Aldrich, Tokyo, Japan) salt was diluted in 1 mL of PBS (1×), making the stock solution. This solution was diluted in a cell culture medium (M199), in a 1:11 ratio. After exposing HUVECs to 50 μg/mL of extracellular histones for 4 h, and washing twice with PBS (1×) solution, the HUVECs were incubated for 3 h with the 1:11 MTT solution. After this time, 100 µL of DMSO was added to each well and incubated in the dark for 10 min with shaking. Finally, the absorbance of the plate was read at 590 nm, using the SpectraMax Plus 384 (Molecular Devices, San José, CA, USA).
The cell death determination was achieved by flow cytometry, using an Annexin V kit (Immunostep, Salamanca, Spain), following the manufacturer’s specifications. Briefly, after treating HUVECs for 4 h with 50 μg/mL of extracellular histones, cells were recovered together with the medium and resuspended in Annexin V buffer and stained with Annexin V/FITC and propidium iodide (PI). Cell viability, apoptosis, and necrosis were analyzed by flow cytometry in a FACS-Verse cytometer (Beckton Dickinson, San Jose, CA, USA) and determined by the Infinicyt software (Cytognos, Santa Marta de Tormes, Salamanca, Spain).

Lymphocytes present in PBMC (BC group) and ascites (ASC and ASC-CA groups) were phenotyped for the identification of their subsets. A flow cytometric-based assay was used according to standard procedures [37 (link)]. Briefly, the cells were mixed with 50 µL of staining solution containing a mix of fluorochrome-conjugated monoclonal antibodies at a 1:50 dilution; anti-CD3 APC-Cy7 (clone SK7), anti-CD4 PerCP-Cy5.5 (clone RPA-T4), anti-CD25 PE (clone M-A251), anti-CD56 PE-Cy7 (clone B159), anti-CD127 Alexa Fluor647 (clone HIL-7R-M21) (BD Pharmingen™), and anti-CD8 FITC (clone OKT8) (Miltenyi Biotec). Cells were incubated for 30 min on ice and protected from light. After the incubation, cells were washed twice with PBS and the final pellets suspended for acquisition in a FACSVerse cytometer using the FACSuite software (Becton Dickinson, San Jose, CA, USA). FlowJo software was used for the data analysis. The lymphocyte population was identified by the FSC and SSC parameters, and then FSC-Area vs. FSC-Height was used to eliminate doublets. Within the lymphocyte populations, the CD3⁺ lymphocyte population was identified by anti-CD3 APC-Cy7. Within the CD3⁺ lymphocytes, CD4⁺ and CD8⁺ populations were distinguished. Within the CD4⁺ population, the T-reg population was quantified by the parameters anti-CD25 PE vs. anti-CD127 Alexa Fluor647.

U937 cells expressing DC-SIGN were seeded overnight on 96-well plates [10] (link). A six point dilution series of protein G–purified mAbs diluted in RPMI 1640 medium without FCS were incubated with a constant amount of DENV-1 05K2916 (EU081234), DENV-2 TSV01, DENV-3 VN32/96, or DENV-4 2641Y08 at an MOI of 1 for 30 min at 37 °C. Plasma or mAb–virus mixtures were then transferred onto the U937-DC-SIGN cells and incubated for 2 h at 37 °C before adding RPMI 1640, 10% FCS. After incubation overnight, cells were stained intracellularly with Abs against NS1 and E protein and analyzed on a FACSVerse cytometer (Becton Dickinson). Data were analyzed using FlowJo software (TreeStar). The proportions of infected cells were plotted against the dilution factor, and the EC50 was calculated with Prism5 (GraphPad Software), applying a three-parameter non-linear curve fit.

Cells were fixed with glutaraldehyde 25% (v/v) in H₂O (Sigma-Aldrich) [final concentration 0.05% (v/v)] and were counted with a Becton Dickinson FACS-Verse cytometer (Beckton & Dickinson, Franklin Lakes, New Jersey, USA). Fluorescent beads, BD FACSuite™ CS&T research beads (Beckton & Dickinson and Company BD Biosciences, San Jose, USA), were used as internal standard to calibrate the instrument. The cytometer shows fluorescence patterns for FITC, PE, PerCP-CyTM5.5 and APC. To count the Halothece sp. PCC 7418 cells, we selected FITC (488 nm excitation, 530/30 nm emission) and PE (488 nm excitation, 576/26 nm emission) combination fluorescence signals which show clearly the population of the cells. A total of 10,000 cells were counted in each sample and the counted cells were expressed as cells μl⁻¹.

After trypsinization, cells were washed twice in cold PBS containing 5 mM EDTA (PBSE) and then resuspended in 500 µL PBSE. For fixation, 500 µL chilled EtOH was added dropwise and the mixture was incubated at room temperature for 30 min. Fixed cells were collected by centrifugation, resuspended in 500 µL PBSE, incubated with 20 µg RNaseA for 30 min at room temperature and subsequently stained with propidium iodide (PI) by adding 500 µL of a 200 mg/mL PI-stock solution. Samples were stored at 4 °C in the dark until counting using a FACSVerse cytometer (Becton Dickinson, New Jersey, USA.).