Cell lab quanta

About 3 mL well⁻¹ of HT-29 cell suspension (5 × 10⁵) was loaded into each well of a 6-well plate and subjected to incubation at 37 °C for 24 h (5% CO₂). After incubation, the growth media were discarded by aspiration and reloaded with 1 mL of phyto-fabricated ZnO-NPs (1 mg) and colchicine (320 µg) before subjecting to incubation in humified conditions. The treated cells were detached by trypsinization and subjected to centrifugation for 10 min at 2000 rpm and repeatedly washed with PBS. Later, the cell pellet was fixed with ethanol (700 µL) and further incubated (for 1 h at −20 °C). Subsequently, the cells were washed twice with PBS (ice cold) by cold centrifugation for 10 min at 4000 rpm. The resultant cell pellet was resuspended in PBS (1 mL) containing propidium iodide (50 mg mL⁻¹), RNase A (50 mg mL⁻¹), and Triton X-100 (0.1%) and incubated for 30 min under dark conditions before analyzing using a flow cytometer (Cell Lab Quanta™, SC, Beckman Coulter, CA, USA).

The cell cycle phase was evaluated 48 h after transfection by flow cytometry. Briefly, cells were detached from the flasks by a trypsin-EDTA treatment, and nuclear isolation medium (NIM-DAPI 10, Beckman Coulter, Fullerton, CA, USA) was added to the cell pellets in order to stain cells. At least 10000 cells were analyzed using Cell Lab Quanta (Beckman Coulter), and FlowJo software was used to assess cell cycle distribution.” (Materials and Method part, page 13).

Cells were seeded on 12-well plates at a density of 3.5 × 10⁴ cells/well. After one day, medium was replaced with DMEM/F-12 containing 0.40, 1.0, and 2.5 μg/mL cisplatin (Yakult, Tokyo, Japan) or 30 and 100 μg/mL 5-FU (Sigma) plus 1.0 μM folinic acid. Cells were imaged each day and attached cells were counted using ImageJ (NIH, Bethesda, MD).
In order to assess the cisplatin sensitivity of re-constructed cybrids, cells cultivated in the presence of 1.0 μg/mL cisplatin for 7 days were double-stained with Hoechst 33342 and propidium iodide, and then imaged with a fluoromicroscope. Alternatively, double-stained cells were treated with trypsin and subjected to a flow cytometric analysis with a Cell Lab Quanta (Beckman Coulter). Hoechst-positive and propidium iodide-negative cells were interpreted as surviving cells.

Cell lines were seeded in 6-well plates (5 × 10⁶ cells per well; volume 5 ml), pre-incubated for 2 h with 600 nM SCH900776, and subjected to treatment with NAs for 4 h, 14 h, 24 h and 48 h. Stimulated CLL cells were cultured as stated in “Stimulation and treatment of CLL cells”. The cells were harvested and fixed in ice-cold 70% ethanol and stored at −20°C. DNA content was analyzed by PI staining using a Cell Lab QUANTA SC flow cytometer (Beckman Coulter) and Cell Lab QUANTA software (Beckman Coulter). 30,000 (cell lines) or 10,000 (CLL) cells were counted per sample.

Since all lineages of DPSCs had already been phenotypically characterized [12 ], we analyzed just a reduced number of CD markers in the 3rd and 7th passages. We analyzed cryopreserved DPSCs for CD29 (TS2/16, BioLegend, San Diego, CA, USA), CD31 (MBC 78.2, Invitrogen), CD34 (581 (Class III), Invitrogen), CD44 (MEM 85, Invitrogen), CD45 (HI30, Invitrogen), CD73 (AD2, BD Biosciences Pharmingen, Erembodegen, Belgium), CD90 (F15-42-1-5, Beckman Coulter, Miami, FL, USA), CD105 (SN6, Invitrogen) and CD271 (ME20.4, BioLegend) using a flow cytometry analyzer (Cell Lab Quanta, Beckman Coulter). The phenotype analysis was done by first detaching adherent stem cells using the 0.05% trypsin-EDTA solution (Gibco), and then we stained them with primary immunofluorescence antibodies conjugated with phycoerythrin (PE) or fluorescein (FITC). Positive cells were determined as the percentage with a fluorescence intensity greater than 99.5% of the negative isotype immunoglobulin control. The expression of surface markers was classified according to previously established criteria by Suchanek and al. [13 (link)]. Classification criteria: <10% no expression, 10–40% low expression, 40–70% moderate expression, and >70% high expression.

After culturing the cells in each conditioning medium for 48 hrs, we detached the cells from the flasks with trypsin-EDTA treatment, and centrifuged the detached cells. Nuclear Isolation Medium of 0.5 ml (NIM-DAPI 10; Beckman Coulter, Fullerton, CA, USA) was added to cells in the pellets. We determined cell cycle phases from 10,000 cells by using FlowJo software (Tree Star Inc., Ashland, OR, USA) by the Cell Lab Quanta (Beckman Coulter) with an excitation at 365 nm and emission at 450 nm for DAPI.