Cells
Mouse neuroblastoma NIE115 cells were purchased from ATCC (Manassas, USA) and were maintained in DMEM (ATCC) with 10% FBS (Invitrogen, Carlsbad, USA).
Antibodies and reagents
A rabbit anti-CDK6 primary antibody (sc-117, used at a dilution of 1:50) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). The secondary goat anti-rabbit IgG Alexa Fluor 660 conjugated antibody (highly cross- adsorbed, used at a dilution of 1:500) was purchased from Invitrogen (Carlsbad, USA). MiR-124-FITC (cat#38507-04, C = 25 μM), scrambled-miR-FITC (cat#99004-04, C = 25 μM), and miRCURY™ locked nucleic acid (LNA) detection probes were purchased from Exiqon (Woburn, USA) and used at a dilution of 1:100. Hybridisation buffer (25% formamide, 15 mM NaCl and 10% dextran sulphate) was prepared as described previously [8].
Staining for miR-124 and CDK6
The NIE115 cells were washed with PBS and permeabilised using the BrdU Flow kit (BD Biosciences, San Jose, USA) according to the manufacturer's instructions. After washing with Perm/Wash Buffer from the BrdU Flow kit, the cells were incubated in Perm/Wash buffer containing 50% goat serum for 20 min, after which the anti-CDK6 antibody was added. The cells were incubated with the primary antibody for 30 min at 20°C (room temperature), washed twice with Perm/Wash buffer, and then incubated with the AlexaFluor 660-conjugated secondary antibody in Perm/Wash Buffer at 20°C for 20 min. After incubation, the cells were washed twice in Perm/Wash Buffer and fixed with 1% paraformaldehyde in PBS for 15 min on ice. After fixation, the cells were washed with PBS and resuspended in hybridisation buffer. The cells were incubated for 30 min at 53°C followed by the addition of miR-124-FITC or scrambled-miR-FITC probes. The cells were incubated with the probes for 1 h at 53°C and then washed twice with 1X saline-sodium citrate (SSC) buffer at 53°C, washed once with 0.1× SSC buffer at 53°C, washed once with PBS at 20°C, and finally resuspended in 1% paraformaldehyde in PBS. The cells were stored at 4°C until analysis.
Transfection with miR-124
5-10 × 106 neuroblastoma NIE115 cells were transfected with 50 nM of a miR-124a duplex that mimics pre-miR-124a (sense 5'-UAAGGCACGCGGUGAAUGCC-3', antisense: 3'-UUAUUCCGTGCGCCACUUAC-5', Invitrogen, Carlsbad, USA), using Lipofectamine 2000™ (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. As a negative control for miR-124, control miRNA (Negative Control#1, sense: 5'-AGUACUGCUUACGAUACGGTT-3', antisense: 5'-CCGUAUCGUAAGCAGUACUTT-3', Invitrogen, Carlsbad, USA) was used. Cells were analysed 48 h post-transfection.
BrdU incorporation assay
Proliferation was assessed by examining bromodeoxyuridine (BrdU) incorporation 14 h after the addition of BrdU to the neuroblastoma cell culture. Analysis of BrdU labelling of cells was performed using the BrdU Flow kit from BD Biosciences (San Jose, USA) according to the manufacturer's instructions.
Quantitative imaging flow cytometry
After staining for miR-124 and CDK-6 and fixation, cells were adjusted to 107 cells/ml and transferred to 500 μl siliconised microcentrifuge tubes (Sigma, St Louis, USA) for analysis on an ImageStream 100 imaging cytometer (Amnis Inc, Seattle, USA). We successfully used the AMNIS cytometer in our previous applications to study the extent of transfection of macrophages with microRNA-124 [5]. The ImageStream was equipped with 488, 658, and 405 nm laser sources with variable laser power (from 20 to 200 mW for the 488 nm laser and from 20 to 90 nm for the 658 nm laser) and a brightfield light source. Files of 10,000-20,000 events were acquired for each sample and 200-500 events were acquired for single fluorochrome controls. Single colour controls were collected first to set the optimal laser power and to avoid saturation of the camera. Additional single colour control files were collected in the absence of brightfield illumination for use in creating the compensation matrix with IDEAS software (Amnis, Seattle, USA). The calculated compensation matrix was applied to all files to correct for spectral crosstalk. The resulting compensated cytometry data were further analysed with the IDEAS software program. Gating of cell events with the area and aspect ratio was used to eliminate debris (low area) and multi-cellular events (large area, high aspect ratio) from further analysis, as has been described before [13]. After defining single, focused cells, the identification of appropriate cell subpopulations was done by analysing the cells labelled for BrdU, CDK6, and/or miR-124. The ratio of the fluorescent pixel intensity to the area of the cell in their respective channels was analysed for miR-124 and CDK6 staining using the IDEAS software.