Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 108 infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.
|Number of pages||6|
|Publication status||Published - 1 Jan 2017|
- cell-defined array
- reverse infection