Quantification of Proliferative and Dead Cells in Enteroids

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

Transcript

This protocol is useful for enteroid isolation in a culture in addition to investigating cell proliferation and the survival in enteroids. Scientists can apply this technique to culture enteroids and to study the effect of drugs and inflammatory cytokines on intestinal epithelial cells in vitro. The implications of this technique extend to water therapy of inflammatory bowel disease.

We apply this method in to find some therapeutical cytokines. This method provides insights into intestinal epithelial proliferation and survival;in addition it may also be used in organoids, cultured around tissues other than the intestine. To isolate intestinal crypts and perform enteroid culture, use tissue forceps and find iris scissors to dissect out approximately eight centimeters of ileum from a euthanized eight week old wild-type mouse.

Use a syringe with a gavage feeding needle to flush the ileum with about 40 milliliters of ice cold Dulbecco's Phosphate-Buffered Saline, then cut lengthwise with scissors and open the ileum. Cut the ileum into small pieces and place them into five milliliters of sterile ice-cold DPBS in a 15 milliliter conical tube. Then rock the sample for five minutes on ice.

Use a pipette controller to aspirate the DPBS and replace it with 10 milliliters of cold buffer one. After rocking for 30 minutes on ice, use the pipette controller to aspirate buffer one and replace with 10 milliliters of cold buffer two. Then shake for two to three minutes by hand at approximately 80 shakes per minute.

After shaking, inspect a twenty micro liter droplet of buffer two contents under a microscope to ensure there are crypts with granular Paneth cells. Filter buffer two contents with a 70 micron sterile cell strainer and collect the filtered buffer in a 50 milliliter conical tube. Pipette 20 microliters of filtrate onto the slide to count the crypts.

Transfer a sufficient volume of the filtered buffer two contents to ensure that there are approximately 500 crypts per well. Spin down at 150 times G for 10 minutes at four degrees Celsius, and then carefully aspirate the supernatant. Resuspend the crypts in 50 microliters of basement membrane matrix per 500 crypts.

Pipette up and down taking care to avoid bubbles. Place a 50 microliter droplet of basement membrane matrix and crypt mix in the center of one well of a 24 well plate. Incubate for 30 minutes at 37 degrees Celsius to polymerize the basement membrane matrix.

After 30 minutes of polymerization, carefully add 600 microliters of minigut media to each well. Return the plate to the 37 degrees Celsius incubator, and observe under a microscope each day, changing the media every two to three days. To passage the enteroids, place the tissue culture plate on ice, aspirate the media, and add one milliliter of cold DPBS to each well.

Use a P1000 tip to pipette up and down until no matrix chunks remain. Pass the sample up through a one milliliter insulin syringe and down into a 15 milliliter conical tube. After spinning down for five minutes at 150 times G and four degrees Celsius, use a pipette to remove the DPBS.

Re-suspend the the enteroids in 50 microliters of basement membrane matrix per well. Place the plate in the 37 degrees Celsius incubator for 30 minutes to allow the basement membrane matrix to polymerize. Then, overlay each well with 600 microliters of ENR media and return the plate to the incubator.

Grow enteroids for five to seven days before passaging them into a new 24 well plate using a splitting ratio of one to two. Add 600 microliters of ENR medium to each well, and then incubate the enteroids for four to five days in the 37 degrees Celsius incubator. Set up the control group of untreated enteroids and the experimental group of enteroids treated with five nanograms per milliliter of interleukin-22.

Prepare at least three replicates for each group. Add 600 microliters of EdU medium to each well of enteroids except one well to use as a negative control for background subtraction. Incubate the plate for two hours in a 37 degrees celsius incubator.

To harvest enteroids from the basement membrane matrix, use a pipette controller to aspirate EdU medium and wash once with DPBS. Then, add one milliliter of DPBS. Use a P1000 pipette tip to pipette up and down until no solid basement membrane matrix chunks remain.

Transfer the sample to a 15 milliliter tube and spin down at 300 times G for five minutes before discarding the supernatant. Add 500 microliters of cell dissociation enzymes and incubate for 15 minutes at 37 degrees Celsius. Then use a P200 pipette tip to pipette up and down and break enteroids into single cells.

Next, add three milliliters of Dulbecco's Modified Eagle Medium containing 10%fetal bovine serum, and repeatedly pipette with a P1000 pipette tip. After centrifuging at 300 times G for five minutes, and aspirating the supernatant, we suspend the cells in one milliliter of DPBS. To perform fixation and permeabilization of cells, first transfer the cell suspension to a one point five milliliter tube, spin down at 300 times G for five minutes, and discard the supernatant.

We suspend the cells in one milliliter of 4%paraformaldehyde and fix for 15 minutes at room temperature. After centrifuging at 300 times G for five minutes, aspirate the supernatant with a pipette tip. Wash the cells once with the DPBS, and spin down again to remove the supernatant.

Resuspend the cells with one milliliter of 0.5%non-ionic surfactant, and incubate for 10 minutes at room temperature before washing the cells with DPBS as before. To detect the EdU, add 100 microliters of reaction cocktail to each 1.5 milliliter tube. Resuspend the cells, and protecting them from light, incubate for 30 minutes at room temperature.

After centrifuging at 300 times G for five minutes, gently aspirate the reaction solution with a pipette tip Add 0.5%non-ionic surfactant penetrant to each tube to wash once at room temperature. Following centrifugation as before, we suspend the cells in one milliliter of DPBS. Filter the resuspended cells with a 40 micron strainer and collect the filtered cells in a 15 milliliter conical tube.

Perform facts on machine detection as soon as possible. Select the appropriate channel and voltage for flow cytometry analysis. For the gating strategy, draw an FSC-A versus SSC-A pseudocolor plot and distribute most of the cells to the visible range of the dot map by adjusting the voltage.

Select the cell population as R1 and exclude the cell debris in the bottom left corner. From the R1 cell population, establish an FSC-A versus FSC-H pseudocolor plot. Set the gate to select single cells designated as R2 and exclude cell clumps.

From the R2 cell population, establish the fluorescence intensity versus cell number plot and use the negative control to set the gate. The region of the fluorescent signal is a positive cell region designated R3.Compare the ratio of these EdU positive cells between the experimental and control groups. Small intestinal crypts were isolated and cultured as enteroids in the basement membrane matrix.

Enteroids started to form buds two days after isolation. On day six, enteroids had many buds with lots of debris in the lumen. Enteroids were ready to be passaged at this stage.

Enteroids were treated with IL-22 for 3 days, after which the synthetic DNA was labeled with EdU in red to indicate cell proliferation. IL-22 treated enteroids displayed an increased number of EdU positive cells. IL-22 increased proliferating cells from 40.1%to 83.5%as analyzed by flow cytometry.

IL-22 treatment also increased the cell death in enteroids indicated by propidium iodide staining in red. IL-22 increased dead cells from 4.9%to 16.2%as analyzed by flow cytometry. During the crypt isolation progress fair shaking is needed after adding buffer to ensure enough crypts are shaken down.

We advise inspecting contents under microscope. Following this procedure, we can also quantify the differentiated epithelials types and enteroids by using specific This method will help researchers to investigate intestinal epithelial differentiation in enteroids. After its development this question paves a way for researchers in the field of gastroenterology to explore the developmental or pathological questions in the cut.

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Journal (Biology)

Journal (Biology)

Quantification of Proliferative and Dead Cells in Enteroids

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