This protocol provides a method to produce several hundred multicellular tumorous spheroids in each well of a multi well plate suitable for automated microscopy. Spheroid produced can be categorized into different size classes, and subcellular information can be extracted from every cell. The technique produces sufficient serums that can be used in direct screening and RNA interference studies.
Dilute ECM basement material in cold phenol red serum free medium using pre chilled tips kept at minus 20 degrees Celsius. I bet 15 microliters of the ECM basement material and medium solution into a 96 well imaging plate. Centrifuge the plate for 20 minutes at 91 times G at four degrees Celsius.
Incubate the plate for no longer than 30 minutes at 37 degrees Celsius. Add trips and EDTA to the cells. Incubate the cells at 37 degrees Celsius at the end of incubation, we suspend the cells in a complete medium containing 10%FBS.
Pipette 10 microliters of the cell suspension, into a hemocytometer counting chamber. Count the number of cells in four quadrants of the chamber. Centra views the cells at 135 times G for four minutes at room temperature.
One this cells are pelleted, aspirate the supernatant and resuspend the cells in a phenol red free complete medium to obtain a concentration of 10 to the six cells per milliliter. Dilute the cells in a phenol red free complete medium. See the cells dropwise in a circular motion in a total volume of 35 microliters per well.
Incubate the cells for one hour at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. At 1.2 microliters of ECM basement material to 23.8 microliters of phenol red free complete medium per well, resulting in a final volume of 60 microliters per well. Incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.
The following day, replace the medium with 100 microliters of fresh phenol red free complete medium. Insert the 96 well plate containing spheroids, into a confocal high content screening microscope. Select the appropriate lasers to excite the floors used.
Acquire the channels sequentially to avoid crosstalk, image the spheroids using appropriate objectives. To analyze the morphology of spheroids, segment the spheroids based on the fluorescently conjugated FLoid and staining. Segment the nuclei based on the hex three, three, three, four, two staining.
Segment the cytoplasm of each cell by the residual hex three, three, four, two, stain that is present in the cell cytoplasm. Calculate different morphological properties of the spheroids including volume, surface area, sphericity and the number of nuclei per his spheroids. Ross has images further to remove spheroids smaller than 75 cubic micrometers and a larger than 900, 000 cubic micrometers.
To analyze a single cell within spheroids, segment the spheroids based on the fluorescently conjugated spheroid staining. Segment the nuclei based on the hex three, three, three, four, two staining. Segment the cytoplasm of each cell by the residual hex three, three, three, four, two stain that is present in the cell cytoplasm.
Segment the lysosomes based on the secondary antibody staining. Calculate different morphological properties of the cells within each spheroid, including the number of lysosomes per cell, cell volume and cell surface area. The figure shows well overviews of three different types of spheroids, including images at different confocal planes.
A volumetric view of the entire spheroids is also shown. The figure shows the pH segmentation process, panel A shows the entire spheroids in a volumetric view. Panel B displays the set segmentation of the cytoplasm, the nuclei and the lysosomes within all cells of a single spheroids.
The cytoplasm and nuclei are segmented based on the hex three, three, three, four, two staining. And lysosomes are segmented based on the anti lab one antibody staining. Panels C shows the same segmented as panel B, but in a three plain view.
Various morphological measurements at the spheroids level can be made. These measurements include calculation of the number of nuclei per spheroids, as well as these Felicity, volume, and surface area of the pH. The typical volume of each cell in the three spheroid types.
As well as the number of lysosomes per cell are shown in this figure. Optimizing the initial cell seating density at the start of SP generation is a critical step. The figure shows that adding too many cells still allows spheroids formation.
However, this can result in the fusion if spheroids. Spheroids produce in this manner can be subjected to transcriptomic and proteomic analysis. This technique is used to quantitatively analyze the uptake of drug delivery such nano particles and assess their toxicity to individual cells in the spheroids.
