TITLE: Live Cell Quantification using Image Analysis
AUTHORS: Minjung Song, Iman Mali, Venkat Pisupati, Florian T Merkle

[DESCRIPTION]
Purpose
This protocol describes image-based quantification of human pluripotent stem cells (hPSCs), which could be adapted for other cell populations that grow in a monolayer. It utilises a membrane-bound stain and requires a high-content imaging system such as a PerkinElmer Oprea Phenix or similar and downstream image analysis pipelines. The suggested dye labels live cells, fluoresces in far red, and shows no evidence of toxicity in hPSCs. This protocol is useful in situations where cell quantification by manual or automated counting becomes rate-limiting, such as when multiple lines are grown for high-throughput differentiation experiments on different genetic backgrounds, or for survival or morphology-based screens in systems where cells have clearly defined cell borders.

[BEFORE_START]
Prepare Staining Solution as described in section 'Materials'.

[STEPS]
SECTION: Cell culture and staining: Cell plating
1. Coat plate with appropriate substrate to prepare cells for plating. 
 

SECTION: Cell culture and staining: Cell plating
2. When plating cells for culture, ensure that they are evenly distributed in the culture well by using a large enough volume of medium (e.g. 500 mL per well of a 24-well plate) and shaking the plate left-to-right and top-to-bottom, avoiding circular motions that might cause cells to gather in the center. Repeat these motions once the plate has been transferred to the incubator shelf and avoid vibrations by gently closing the incubator, and ensure incubator shelves are level.
SECTION: Cell culture and staining: Cell staining
3. Once cells have reached approximately 80% confluence, aspirate medium and wash once with DPBS then add a standard volume of staining solution (e.g. 500 µL for a well of a 24-well plate) to label the plasma membrane. 
 

SECTION: Cell culture and staining: Cell staining
4. Incubate cells for 10 min at 37 °C.
SECTION: Cell culture and staining: Cell staining
5. Wash cells two times with DPBS. (For a 24-well plate, 500 µL per well was applied).
SECTION: Cell culture and staining: Cell staining
5.1. Wash cells with DPBS. (1/3)
SECTION: Cell culture and staining: Cell staining
5.2. Wash cells with DPBS. (2/3)
SECTION: Cell culture and staining: Cell staining
5.3. Wash cells with DPBS. (3/3)
SECTION: Cell culture and staining: Cell staining
6. After final washing, add culture media. Plates can be imaged immediately, or returned to the incubator and imaged within 2 hours of staining.
SECTION: Imaging
7. 
 

SECTION: Imaging
8. Seal the wells of the plates with sterile B seal film and bring the plate to the imaging facility. While wearing gloves, carefully wipe the bottom of the plate with a clean tissue to ensure that no dust, fingerprints, or media interfere with imaging.
SECTION: Imaging
9. For imaging cells stained with CellMask Deep Red plasma membrane dye, use a 647 nm laser at approximately 50% power setting and an exposure time of approximately 200 ms, to be optimised by the user. We have found it helpful to take a stack of 3 optical sections separated by 0.8 microns and starting at the surface of the plate where cells are attached with 20x Water objective. The dye stains the plasma membrane, and the images should clearly reveal the cell borders so that images can be readily segmented for quantification.
SECTION: Imaging
10. Image all wells to be quantified. It should be possible to image the entire plate in less than 30 minutes, in which case it can be performed at 37 Room temperature.

SECTION: Imaging
11. Return the plate to a biosafety cabinet after wiping it well with 70 %. Carefully remove B seal film and return the plate to the incubator while awaiting results of image quantification.
SECTION: Image analysis
12. 
 

SECTION: Image analysis
13. In input images, select basic flatfield correction and maximum projection. (For max projection analysis, make sure all z-planes are selected).
SECTION: Image analysis
14. Invert maximum projected images.
SECTION: Image analysis
15. Remove noise from the background (e.g. with a sliding parabola filter) with settings to be optimised by the user.
SECTION: Image analysis
16. Segment cells to enable quantification. The exact parameters will depend on the cell type and density. For hPSC quantification at approximately 80% confluence with Harmony, we found that the ‘Find nuclei’ function (method B and M) worked well. Other parameters such as threshold, size and splitting sensitivity will need to be adjusted to refine the identification. 
 

SECTION: Image analysis
17.