Scepter 2.0 cell counter

Commercially available HPL (UltraGRO^TM, Helios BioScience, Atlanta, GA) was used for ASC culture supplement. ASCs were cultured in DMEM-high glucose (DMEM-HG; Hyclone) supplemented with 10% FBS, 5% HPL, 2% HPL or 1% HPL and subjected to flow cytometry analysis to determine the cell surface antigen expression. The cells were incubated with the following antibodies: human monoclonal antibodies against CD31 (BD Pharmingen, San Jose, CA, United States), CD44, CD34, CD73, CD90, and CD166 (all from BioLegend, San Diego, CA, United States). The samples were analyzed using a flow cytometer (FACSVerse; BD Biosciences, Franklin Lakes, NJ, United States) in which 10,000 cells were counted per sample. Positive cells were determined as the proportion of the population with higher fluorescence than 95% of the isotype control. Moreover, ASCs were seeded in 10 cm culture dishes and supplemented with 10% FBS, 5% HPL, 2% HPL, or 1% HPL. After 7 days, the cells were lifted, counted with Scepter 2.0 Cell Counter (Millipore, Billerica, MA, United States), and the population doubling time was calculated by the following formula:
N₀ is the seeding cell number at the beginning of the incubation time (1.6 × 10⁴ cells per dish). Ne is the cell number at the end of the incubation time.

Citrated blood samples were incubated with dextran (3%) for 45 min in order to isolate human polymorphonuclear leukocytes (PMNs). The supernatant was dropped over Ficoll-Hypaque (GE Healthcare, Barcelona, Spain) and centrifuged for 25 min at room temperature at 650 g. Lysis buffer was added to the erythrocytes remaining in the pellet, which was incubated at room temperature for 5 min and then spun at 240 g for 5 min. Leukocytes were washed twice and resuspended at 37 °C in Hanks’ balanced salt solution (HBSS; Sigma Aldrich, MO). Scepter 2.0 cell counter (Millipore, MA, USA) was employed to count cells. Each cellular suspension was divided into two samples, one of which was treated for 30 min with 100 nM of SS-31, and the other with SS-20 in identical conditions (these concentrations did not affect the viability of the cells).

Cells were plated at 40,000 per well of 12- well plate, and cultured with growth medium. After 4 days, cells were dissociated in trypsin-EDTA (0.05%, Life Technologies). Cell number was counted by Scepter™ 2.0 Cell Counter (Millipore).

Single-cell analysis was performed at the indicated days of culture at the air-liquid interface. To obtain a single-cell suspension, cells were incubated with 0.1% protease type XIV from S. griseus (Sigma-Aldrich) in supplemented HBSS for 4 h at 4°C. Cells were gently detached from Transwells by pipetting and then transferred to a microtube. Fifty units of DNase I (EN0523 Thermo Fisher Scientific) per 250 µl were directly added and cells were further incubated at room temperature for 10 min. Cells were centrifuged (150 g for 5 min) and resuspended in 500 µl supplemented HBSS containing 10% FCS, centrifuged again (150 g for 5 min) and resuspended in 500 µl HBSS before being mechanically dissociated through a 26 G syringe (four times). Finally, cell suspensions were filtered through a 40 µm porosity Flowmi Cell Strainer (Bel-Art), centrifuged (150 g for 5 min) and resuspended in 500 µl of ice-cold HBSS. Cell concentration measurements were performed with a Scepter 2.0 Cell Counter (Millipore) and Countess automated cell counter (Thermo Fisher Scientific). Cell viability was checked with a Countess automated cell counter (Thermo Fisher Scientific). All steps except the DNase I incubation were performed on ice. For cell capture using the 10× genomics device, the cell concentration was adjusted to 300 cells/µl in HBSS, aiming to capture 1500 cells for HAECs and 5000 cells for MTECs.

For erythrocyte isolation, whole blood was obtained from mice in citrate-phosphate-dextrose buffer (16 mM citric acid, 90 mM sodium citrate, 16 mM NaH₂PO₄, 142 mM dextrose, pH 7.4) in a ratio of 1/10 of the blood volume. Then, the mixture was transferred to a fresh 1.5-ml Eppendorf tube. After gentle pipetting, the samples were centrifugated at 200×g for 20 min at room temperature. After removing the top and middle layers, 90% of the volume of the bottom layer was transferred to a fresh 1.5-ml Eppendorf tube. For washing, 1 ml of cold 1X phosphate-buffered saline (PBS; Gibco, #10010-023, USA) was added, mixed gently, and centrifuged at 2000×g for 20 min. The supernatant was discarded and the wash process was repeated three times. The erythrocytes isolated were prepared for the next experiment. For erythrocyte cell counting, the Scepter 2.0 Cell Counter (Millipore, #PHCC00000) and Scepter Sensors (Millipore, #MER-PHCC60050) were used.

To determine the optimal concentration of 3-deazaneplanocin A (DZNep) and suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich St. Louis, MO, USA) in prostate cancer cell lines, DZNep and SAHA were dissolved in dimethyl sulphoxide (DMSO, Sigma-Aldrich, St Louis, MO, USA). Final DMSO concentration did not exceeded 0.1%. The same concentration of DMSO was used as a control for these experiments. We measured cell viability using a Scepter™ 2.0 Cell Counter (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s standard operating procedures. To identify the demethylating and deacetylating effects of DZNep and SAHA, DU145, LNCaP and PC3 cells were distributed in six-well culture plates at a density of 0.5×10⁵ cells per well. After 24 hours, cells were treated with increased doses of DZNep and SAHA using 10 μl DMSO for respectively and gradually 24, 48 and 72 hours. After treatments, cells were washed with phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA, USA), trypsinized, harvested and the number of viable cells was counted. Viable cells were presented as a percentage of the untreated cells control and IC₅₀ is done by linear interpolation between concentrations just above and beneath 50% inhibition in the response dose curve.