Nucleocounter system

After digestion of spheres to single cells, the cell number and viability was determined using the Nucleocounter system according to the manufacturers' instructions (Nucleocounter, Chemometec, Allerod, Denmark). The method allows detection of non-viable and viable cells in cell suspensions using propidium iodide (PI). The sphere viability was also investigated in intact spheres with fluorescein diacetate (FDA) and PI. Spheres were spun down by centrifugation and incubated for 3 minutes in 0.02 mg/ml FDA and 0.1mg/ml PI at room-temperature. Labeled cells were imaged using an Olympus IX81 inverted fluorescence microscope. Images were acquired using Olympus soft imaging Excellence software. Post processing of the images was done using the ImageJ software (http://imagej.nih.gov.com).

Cells were treated with different doses of romidepsin and/or JQ1 for up to 96 h. Cell proliferation and cell viability was measured using the Guava ViaCount reagent (Merck Millipore, Darmstadt, Germany) or the NucleoCounter system (Chemometec, Allerod, Denmark). Metabolic activity of cells was measured using the WST-1 method (Roche, Basilea, Switzerland) which allows the quantification of the number of viable cells by the cleavage of tetrazolium salt WST-1 to formazan dye. Romidepsin and JQ1 combination effects were determined using the combination index (CI) values, analyzed by the Chou-Talalay method using CompuSyn Software^{63 (link)}.

The ectopic expression of miR-451a was obtained in NIM1 and TPC1 cells by the transfection of miR-451a synthetic miRNA mimic (PM10286 Applied Biosystems) at 100 nM by siIMPORTER Transfection Reagent (Millipore, Billerica, MA); FAM-labeled Negative Control#1 (AM17121 Applied Biosystems) was transfected as negative control. Following transfection both cell lines were evaluated for cell number and proliferation. Cell number was assessed by NucleoCounter system (ChemoMetec A/S, Denmark) as previously described [34 (link)]. Cell proliferation was assessed by crystal violet assay. Briefly, at the indicated time points transfected cells were first fixed with 10% formalin for 20 min and then stained with 0.1% crystal violet (Sigma-Aldrich, MO, USA) for 30 min. After stain removal and PBS washes, the dye was solubilized with 1% SDS and the absorbance was measured at 570nm by a microplate reader (TecanUltra, Tecan Trading AG, Switzerland).

Each patient underwent bone marrow aspiration from
the posterior superior iliac crest while in the right or left
lateral positions under local anesthesia. The MSCs were
prepared from bone marrow sample (100 ml) according to
current good manufacturing practice (cGMP).
Mononuclear cells (MNCs) were isolated from the BM
samples by density gradient with a Ficoll Paque open
system (Lymphodex, Inno-Train, Germany). Next, the
MNC layer was isolated and washed in PBS buffer (Milteny
Biotech GmbH, Germany). Cell counts and viability were
assessed with trypan blue staining and confirmed by a
NucleoCounter® system (ChemoMetec, Denmark). MNCs
(1×10⁶/cm²) were placed in Millicell® HY T-600 culture
flasks (Merk, Germany) and cultured under standard
conditions in 1X MEM alpha medium (Gibco, Germany)
and fetal bovine serum (FBS, Gibco, Germany). Flasks
were incubated under defined conditions of 5% CO₂ and
37°C. All non-adherent cells were removed by changing
the culture medium after 3-4 days. This process was
repeated every 3 days. After 1 to 2 passages, the 90%
confluent MSCs were harvested by the application of
0.25% trypsin in 0.1% Ethylenediaminetetraacetic acid
(EDTA). Cell viability was evaluated by trypan blue
staining as well as the NucleoCounter® system. Next, we
suspended MSCs (2×10⁶ cells/kg) in 5 ml of 0.9% sodium
chloride that contained 2% human serum albumin.