Cytomics fc500

PBMCs were treated with 50 μmol C4 for 1 h and stimulated with 1 μg/mL H37Rv or 10 ng/mL LPS for 7 days. Subsequently cells were restimulated with 200 μL RPMI supplemented with 10% serum, Golgi-plug inhibitor (GPI Brefeldin A; 1 μg/mL, BD Pharmingen), PMA (phorbol 12-myristate 13-acetate; 50 μg/mL, Sigma-Aldrich), and ionomycin (1 μg/mL, Sigma-Aldrich) for 4–6 h at 37°C and 5% CO₂. Cells were then washed with PBA (PBS 1% BSA (albumin from bovine serum)) and stained extracellularly for 30 min with CD4-PeCys7 (ITK) for T-helper 17 (Th17) cells at 4°C. Next, cells were washed and permeabilized by fix and perm buffer (eBioscience) according to the manufacturer's protocol for 45–60 min at 4°C. Finally cells were washed and resuspended in 300 μL PBA to be measured using the Cytomics FC500 (Beckman Coulter) (n = 8).
Cell death was measured by staining PBMCs with Annexin V-FITC (BioVision) and Propidium Iodide (PI) (Invitrogen Molecular Probes). Cells were incubated in the dark on ice with Annexin-V staining solution (RPMI supplemented with 5 mM CaCl₂ and 0.1 μL/mL Annexin-V) for 15 minutes. Subsequently PBMCs were stained with PI for 5 minutes. Cells were measured with the Cytomics FC500 (Beckman Coulter, Woerden, Netherlands), and data were analysed using CXP analysis software v2.2 (Beckman Coulter) (n = 3 to 5).

KYSE30 and KYSE180 cells were plated into 6-well plates (2 x 10⁵ cells/well) in antibiotic-free medium after being transfected with sh-HOTAIR, sh-HK2, sh-NC, hsa-miR125a-5p mimics, hsa-miR-143 mimics or NC mimics for 48 h. Cells were collected for apoptosis analysis, washed twice and stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI, BD Bioscience) using a FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s manual. Apoptotic cells were analysed using flow cytometry (CYTOMICS FC 500, Beckman Coulter, Miami, FL). We calculated the apoptotic cells according to the manufacturer's instructions as follows: the fraction of cells in the upper right quadrant (representing late apoptotic cells) and the fraction of cells in the lower right quadrant (representing early apoptotic cells).
The KYSE30 cells for cell cycle analysis were suspended in 70% chilled ethanol and washed with cold PBS prior to staining with PI. Cell cycle distribution was measured using flow cytometry (CYTOMICS FC 500, Beckman Coulter, Miami, FL). The percentage of cells in the G0-G1, S, and G2-M phases were counted and compared. All experiments were conducted in triplicate.

CD64 measurement was performed on the same blood sample collected routinely for CBC at admission (1.0 mL), in most cases without the need for additional venipuncture. In some instances, a blood sample was obtained specifically for CD64 determination.
For method #1, phosphate-buffered saline-diluted whole blood (50 μL) was incubated for 15 min at room temperature with a combination of CD64 FITC- (fluorescein isothiocyanate-) clone IM1604u (Beckman Coulter) and CD45 PE- (phycoerythrin-) clone IM2078 (Beckman Coulter). After lysis of red blood cells, samples were washed and fixed.
CD64 was assessed in neutrophils separated by marking with CD45, using the CD45x Side Scatter graphic (SSC). The result was assessed by calculating the ratio between the Mean Gene Expression (MnX) obtained from the patient and the MnX obtained from the control. The result expressed in log scale was directly related to the antigenic density of the CD64 monoclonal antibody on the cell surface.
The readings were done on Cytomics FC500 (Beckman Coulter).
For method #2, the commercial kit Leuko64 (Trillium Diagnostics, LLC) was used and readings were done on the Cytomics FC500 (Beckman Coulter) equipment. The results were assessed with the QuantiCALC software. According to the instructions in the kit, the expected index in normal subjects is PMN CD64 ≤ 1.00.

We chose the 25 LS MACS separator columns in order to get 10⁸ cells as the maximum number of labeled cells and 2 × 10⁹ maximum number separators of total cells. The column was placed in the magnetic field and was prepared by rinsing through 2 × 3 mL of PBS each column. Then the cell suspension was applied onto the column. We collected the unlabeled cells that passed through and the column was washed with 3 × 3 mL of buffer. The total effluent was collected (this was the unlabeled cell fraction). We repeated the washing step by adding buffer three times. After the washing step was finished we removed the column and placed it on a 25 mL Falcon tube. A 5 mL buffer was pipetted into the column and immediately we flushed out the magnetically labeled cells by firmly pushing the plunger into the column.
The total volume of 5 mL ADSC (Figure 5) was collected in the 25 mL Falcon tube and was placed in ice before the infusion in our animal models.
A Cytomics FC500 by Beckman Coulter was used with CXP software for the Cytomics FC500 flow cytometry system version 2.2. We used 500 μL from the total volume of 5 mL ADSC in order to count the total number of cells and their viability, by adding 10 μL of 7-AAD for viable cells, using flow cytometry. A total of 100,000 cells/mL were isolated. Sample analysis was completed typically within 10 minutes.

The cells (2 × 10⁵ cells/well) were seeded in 6-well plates and incubated for 24h. On the following day, the cells were treated with the designed concentrations of 4HNE or 4HNE-Trx1 adduct for 12h. Subsequently, the cells were collected by centrifugation for 5 min at 2500 rpm, followed by washing with PBS for 3 times. The cells were stained with both annexin V-FITC and PI for flow cytometric analysis. The percentage of apoptotic cells was measured using a flow cytometer (Cytomics FC 500, Beckman, USA).
Alternatively, the cells were stained with 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) and incubated at 37°C for 30 min in the dark. Then, the cells were washed with PBS for 3 times and applied to determine ROS level using a flow cytometer (Cytomics FC 500, Beckman, USA).