Cell Cycle Analysis: An Approach to Study Cell Cycle Regulation of miRNA-transfected Lung Cancer Cells

1. miR-143 and miR-506 transfection

CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat while performing the described experiments. When required, use the biosafety cabinet with the cabinet fan on, without blocking the airways or disturbing the laminar airflow. Always set the protecting glass window to the appropriate height, as described by the manufacturer.

1. Seed NSCLC A549 cells in a T25 cm² flask/6/96 well plate in DMEM/F12K media supplemented with 10% FBS and 1% penicillin-streptomycin (culture media) in a tissue culture hood and incubate overnight at 37 °C with 5% CO₂ in a tissue culture incubator.
2. Suspend miR-143 and/or miR-506 mimics, or scramble siRNA with transfecting agent (2.4 µg of miR were mixed with 14 µL of transfecting agent; see Table of Materials) in 500 µL of transfection media and 1.5 mL of serum and antibiotic-free DMEM/F12K media at a final miRNA concentration of 100 nM. miRNA amount may require optimization on different cells and concentrations. Appropriate approaches include the transfection of cells with increasing concentrations of miRNA (i.e., 50-200 nM) and evaluation of expression downregulation of the genes of interest.
3. Remove culture media from flask/plate and wash once with 1x PBS.
4. Add miRNA/scramble-transfecting agent complexes and incubate at 37 °C with 5% CO₂ for 6 h (flask size defines the added incubation volume).
5. Replace the media with 4 mL of culture media and incubate cells for 24 h and/or 48 h.
6. Harvest transfected cells by trypsinization, by adding 1 mL of trypsin-EDTA in each T25 cm² flask, incubate for 5-10 min at 37 °C, and add 3 mL of culture media to harvest the cells. Place the contents of each flask into a separate, marked 15 mL tube. Work in a tissue culture hood.
7. Centrifuge at 751 x g for 5 min and remove the supernatant.
   NOTE: Caution is required during supernatant removal, as agitation of the tube may cause loss of cells.
8. Add 2 mL of 1x PBS and centrifuge for 5 min at 751 x g.
9. Repeat steps 7 and 8 once to remove any traces of media and supernatant.
   NOTE: At this stage, sample tubes can be stored at -80 °C or can be used immediately.

2. Cell cycle analysis

1. Seed 5 x 10⁵ cells for each sample in a T25 cm² flask and perform a transfection according to the protocol described in section 1, steps 1-5.
2. After 24 h and 48 h, then harvest the cells by trypsinization.
3. Transfer the cell suspensions to 15 mL sterile tubes and label them properly.
4. Centrifuge samples at 751 x g for 5 min and discard the supernatant.
5. Add 2 mL of ice-cold 1x PBS, vortex, and centrifuge at 751 x g for 5 min. Discard the supernatant.
6. Repeat the washing step with 1x PBS to remove residual media.
7. Resuspend and break the pellet by adding 200 µL of ice-cold 1x PBS by pipetting.
8. Fix the cells by adding 2 mL of 70% ice-cold ethanol dropwise to the tube while vortexing gently.
9. Incubate tubes for 30 min at RT and place the tubes at 4 °C for 1 h.
10. Remove tubes from 4 °C and centrifuge at 751 x g for 5 min.
11. Add 2 mL of ice-cold 1x PBS, vortex, and centrifuge at 751 x g for 5 min. Discard the supernatant.
12. Add 500 µL of 1x PBS with propidium iodide (50 µg/mL) and ribonuclease A (200 µg/mL).
13. Incubate for 30 min at RT while protecting the samples from light.
14. Acquire data on flow cytometer. Use forward vs. side scatter (FSC vs. SSC) to select the main population of cells, excluding debris at the bottom left corner of the FSC vs. SSC density plot and cell clusters at the top to top-right side of the FSC vs. SSC density plot.
15. Analyze data for identification of cell populations per cell cycle stage with appropriate software.