TITLE: A fast and simple fluorometric method to detect cell death in 3D intestinal organoids
AUTHORS: Konstantin J. Bode, Stefanie Mueller, Matthias Schweinlin, Marco Metzger, Thomas Brunner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes a novel fluorometric method for the quantitative measurement of specific organoid cell death. Organoids are stained simultaneously with the cell impermeable nuclear dye propidium iodide and cell permeable Hoechst 33342. While Hoechst allows in-well normalization to cell numbers, propidium iodide detects relative proportion of dead cells independent of hydrogel. Measurement and analysis time, as well as usability are drastically improved in comparison to other established methods. Parallel multiplexing of this method with established assays measuring mitochondrial activity further enhances its applicability in personalized medicine and drug discovery.</div></div>

[STEPS]
?. [Preparing organoids for staining]
Grow murine intestinal organoids as described, preferably in clear 96-well plates.
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Check effects of treatment — morphological differences in treated vs. untreated organoids.
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Treat organoids with Staurosporine or other substance for at least  to ensure disruption of plasma membranes.
Inspect organoid growth and integrity microscopically before starting treatment.
Use technical triplicates at minimum and include negative (untreated) and positive (with Staurosporine) controls.
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Check fluorescence with microscope.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Start plate reader and measurement software (Tecan).
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Remove the lid of the plate and set up the plate reader with the following sub-steps.
Measurement can be performed at  , as it is an endpoint assay.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Set measurement from top position.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Gain must be set using the wells with the highest cell death (Propidium Iodide) and the lowest cell death (Hoechst 33342). The values should fall between 115 and 160 (positive and negative control).If measurement values do not fall within the range, readjust gain and remeasure.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Allow the plate reader to determine the optimal Z-position automatically from the corresponding wells. Check for values between 1.5x106 and 1.6x106 µm. (This can take up to . )
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Measure all wells for Propidium Iodide fluorescence and wait  before measuring Hoechst 33342 fluorescence. Measure fluorescence of each well with 25 flashes and an integration time of 20 µs. Lag and settle time should be set to 0 seconds. Use 4x4 measurements per well, with a border of 1mm around measurement points (measurement points can be increased to counter uneven organoid distribution at the cost of measurement speed).
Excitation and emission wavelengths for PI are 535 nm and 617 nm, respectively. For Hoechst, 361 nm and 486 nm should be used as excitation and emission wavelengths.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Calculate Propidium Iodide/Hoechst 33342 (PI/Hoechst) ratio using relative fluorescence units. \frac{PI}{H}ratio = \frac{RFU(PI)}{RFU(Hoechst)}
?. [Preparing organoids for staining]
Plate 200-300 crypts per well in   Matrigel or BME, incubate for    at , and cover crypts dropwise with  complete murine intestinal organoid growth medium (described in Materials section).
8 µl
[5% CO2]
[pre-warmed]
Depending on whether starting with freshly isolated crypts or thawed organoids, determine timepoint/duration of treatment and analysis.
?. [Preparing organoids for staining]
Allow cultures to grow for 3 days at   before starting cell death treatment.
[5% CO2]
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Stain for  at .
[5% CO2]
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Replace staining medium with  phenol red-free medium.
[pre-warmed, fresh]
?. [Staining of organoids with Propidium Iodide & Hoechst 33342]
Remove treatment medium and replenish organoids with pre-warmed staining medium with final concentration  Propidium Iodide & Hoechst 33342.
?. [Quantification of Propidium Iodide & Hoechst 33342 fluorescence]
Use PI/Hoechst ratios to quantify treatment-specific organoid cell death by dividing each sample with the positive control (e.g. containing Staurosporine), multiplying the value by 100, and then subtracting the PI/H ratio of the untreated samples.  treatment\; specific\; organoid\; cell\; death\; [\%] = \frac{x(sample)}{z(STS)} \times100 - y(ut)