The cell line chosen for the screening experiment is of key importance in the assay development phase. Conditions for most cell lines need to be optimized with regards to assay length, phenotypic readout and growth behavior.
We routinely run large-scale RNAi screens in a number of different Drosophila and human cell lines.
- Luminescence-based reporters are used to quantify cell viability or target gene expression. For measuring cell viability the intracellular ATP level (e.g. CellTiterGlo, see protocols section) is used as indicator. When applied to measure taget gene expression, luciferase, fused to the promotor of the target gene, is transiently or stabely transfected into cells. Usually, the intensity of emitted luminescence light is measured on single well basis by plate readers upon cell-lysis and addition of luciferin (see protocols section).
RENILLA USE COELANTERIZINE AND DOES NOT USE ATP AND LUCIFERIN! Please fact check all entries.
Fluorescence reporters are used to quantify cell morphology and viability, cell cycle, homeostasis as well as biophysical properties and kinetics of cellular processes.
- Nucleus stain. DAPI or Hoechst stain is performed for counting cells per well (proliferation/viability) and for cell cycle analysis using plate cytometry or fluorescence microscopy. The cell cycle is analysed by measuring the total emitted fluorescence intensity per nucleus and subsequent gating from the distribution of cells in one well or all wells of a 384-well plate. See graph
- Cytoskelleton. Tubulin and actin are visualized using fluorescence-labled antibodies. Cytoskelletal stains are used for image segementation (separation and identification of single cells in an image or well) and observation of cell morphology (phenotypic profiling).
- Cytoplasm. Cytomplasmic stain is achieved by loading the cell with a cell-permeant dye (e.g. AM esters) or by transient or stable expression of recombinant GFP variants. This method is choosen for normalization purposes in combination with luminescence reporters or for kinetic measurements of cellular processes.
- Organelles. Sub-cellular compartments such as endosomes, lipid droplets, … are stained using loadeble dyes, antibodies or transfected GFP variants
- Recombinant GFP variants. GFP variants fused to target genes enable quantification of kinetics and localisation of highly conserved signaling pathways relevant in a vaiety of diseases.
Most of the mentioned reporters can be combined in one experiment and serve in a so called multiplexing/barcoding experiment.
- The plate type strongly influencences cost, replicability and the throughput of the screening. Most commonly used plate types for high-throughput screening are 96- and 384-well plates. Established protocols are optimized for 384-well plates as the required material is reduced by up to a factor of four and the throughput is increased four times, compared to 96-well plates.
- Multititer plates often display positional edge effects which can be minimized using approriate techniques and instruments (see section trouble shooting). Positional effects should always be considered when setting-up an assay using multititer plates.
The type of readout depends on the indicator to be used in the assay which is usually either a luminescence- (e.g. Renilla- or Firefly-luciferase) or a fluorescence-based reporter (e.g. GFP-variants or loadable fluorescence dyes such as AM esters).
- Plate reader (Mithras plate reader): The plate reader measures total luminescence or fluorescence signals emitted by all cells of a well and is usually used for quantifying cell viability and the activity of signal transduction pathways.
- Plate cytometer (Acumen eX3): The Acumen eX3 measures fluorescence signals on single-cell basis and is used for counting cell number per well and for cell cycle analysis (e.g. reading the intensity of Hoechst-stained nuclei) as well as for measuring protein enrichment, co-localisation and translocation.
- Fluorescence microscopy (BD Pathway HT855): The fluorescence microscope detects fluorescence signals (multiple wavelengths) of fluorescence-labeled structures such as the cytoskelleton and the nucleus but also cellular (cytoplasma) and sub-cellular compartments (organelles) and is used for functional sudies and for quantifying morphological phenotypes (morphmetry).