The aim of the following procedure is to demonstrate how to sustain the lysosomes of two types of samples, mouse embryos and differentiated stem cells with lyo tracker dye to detect patterns of cell death. To prepare embryo samples, embryos are first surgically isolated and placed into a dish to prepare differentiated cells. Embryonic stem cells are used to create embryo bodies.
These embryo bodies are cultured in suspension and then grown as a monolayer on a chamber slide. The embryos all sell monolayer can be incubated in ot tracker red and are then rinsed and fixed samples can then be photographed to examine and quantify the staining results are obtained that show Liso Tracker. Staining is a simple procedure that can be used to assay program cell death in embryos and cultured cells.
The main advantage of this technique over existing methods like tunnel analysis is that because OT tracker dye is a small molecule, thick tissue can be assayed quickly and easily. Though this method can provide insight into patterns of programmed cell death in mouse embryos and differentiated cultures, it can also be applied to other tissue types such as chicken embryos and differentiating adult stem cells. Pair young female mice with a stud male and on following mornings, check for a vaginal plug, which indicates the mating has occurred.
Noon on the day that the vaginal plug appears is effectively 0.5 days. Post cotus on the embryonic day of interest, euthanize the female according to approved protocols and remove the uterus to a 10 centimeter Petri dish with Hank's BSS and rinse away any blood. Separate the embryos and remove them from the uterus.
Remove the extra embryonic membranes and do at least one rinse with Hank's BSS to remove extraneous tissues and blood. Keep the dissected embryos on ice while cleaning. The others.
Prepare five milliliters of Liso tracker embryo staining solution. This is a sufficient volume for one or two embryo collections. Gently mix the solution to disperse the dye, evenly the then transfer the embryos to the solution and incubate them at 37 degrees Celsius for 45 minutes.
After the incubation, gently rinse the embryos in Hank's BSS four times for five minutes each wash. It is important to do the rinses gently so as to not damage the tissue. Remove the liquid, slowly paying close attention to where the delicate embryos are, and to always leave a portion of the liquid behind covering them.
Then fix the embryos in 4%para formaldehyde overnight the next day. Rinse the embryos once in Hank's BSS for 10 minutes to remove the fixative. Now dehydrate the tissues through a methanol series to eliminate background staining.
This step improves the signal to noise ratio during imaging prior to storage. If needed, collect a tissue sample from each embryo for genetic analysis. Later, rehydrate the tissue sample prior to genotyping.
Separate the embryos into individual tubes with labels that match the tissue sample. Now it is possible to store the isolated embryos indefinitely in methanol of minus 20 degrees Celsius. Protected from light.
Begin by preparing embryo bodies via the hanging drop method. With a starting density of 500 cells per 20 microliter drop after three days, transfer the embryo bodies to suspension cultures on 10 centimeter non-adherent Petri dishes. Culture them for five days, changing the media every two days after five days.
Transfer the embryo bodies to gelatinized eight chamber slides, placing one or two embryo bodies in each chamber. Culture the embryo bodies for another 10 days. Changing the media every other day after 10 days, gently aspirate the existing media and add 300 microliters of Lyo tracker cell staining solution to each chamber.
Be careful to manually pipette out the media placing the pipette tip in the corner of the chamber. This way, you won't accidentally remove the, still attaching every body from the glass surface. When adding back media again, place the pipette tip in the corner of the chamber and add the media back very slowly.
Incubate the chambers at 37 degrees Celsius for 15 minutes. Then rinse them twice with DPBS for five minutes per rinse. Now fix the cells in 4%paraldehyde for 15 minutes at room temperature.
Then remove the fixative with DPBS in two five minute rinses. To mount the embryos, place them in a deep depression glass slide or small Petri dish infector shield. Prepare the cell samples for microscopy by aspirating the DPBS.
Remove the chamber from the slide and allow it to air dry for about five minutes. Cover with foil to protect the fluro. Then add an aqueous mounting medium such as vector shield with dappy and place a cover slip on top.
Now the slide has been mounted and is ready for imaging. Visualize the samples with a dissecting or compound microscope outfitted with a rod, domine or Texas red filter. The staining is typically quite bright, so long exposures are usually not necessary in manual mode.
Take digital photos of all the samples under the same exposure conditions. To quantify the images, open them in Adobe Photoshop or a similar imaging program. To determine the amount of lyo tracker staining, select the red channel.
Note the pixel count for the entire image as revealed by the histogram function. Next threshold, the image such that the red staining is now represented by white pixels. This converts the image to black and white.
Pick a threshold level and use this level for all images in the set. Select the white pixels using the color range tool and use the histogram function to obtain the total white pixel count. Use the blue channel to count the number of nuclei in the field.
It can be useful to keep a record of the count by annotating the image, record the values and use them to calculate the average staining level per cell. Repeat this analysis with the same threshold setting for all the remaining images. To sample ly o tracker was used to stain sonic hedgehog mutant embryos.
Since ly O tracker is a small, highly diffusible molecule areas deep within the meen of a whole embryo as stained at 10.5 days post cotus programmed cell death or PCG was greatly increased in sonic hedgehog null mutant embryos, particularly in the limb and so might regions. Limb buds were imaged in hole mounts at higher magnification to assess the overall pattern. Dissected portions were also sectioned with a vibrating microtone.
The arrowhead points to increased staining in the sonic hedgehog null distal tip in sections obtained with a cryostat tunnel staining was used in conjunction with OT Tracker in these slices, although there is not a strict one-to-one correlation between lyo tracker and tunnel staining, the general regions of PCD overlap considerably under higher magnification. It is possible to see puncture that stained positive with the tunnel assay, but not the otra die puncture that stain only with the lyra die and puncture that have overlapping staining. After 10 days of embryo body culture cells differentiated into a variety of cell types, including neurons, muscle cells, and cardiomyocytes.
In the normal culture of differentiated ES cells, there are a few cells that underwent PCD as detected with lyo tracker and tunnel assays. Next cells were treated with hydrogen peroxide to induce oxidative stress cell death. A low dose of hydrogen peroxide increased both lyo tracker and tunnel staining compared to no treatment.
Similar to the results in the embryo, both stains labeled common and unique cells. A high dose of hydrogen peroxide resulted in cell shrinkage and more widespread OT tracker and tunnel positivity. For comparison, serum starvation was used to induce autophagy after one hour of serum starvation.
OT tracker staining increased. However, tunnel positivity remained at normal levels. Thus, while reer is easy and useful, it is important to follow up with additional assays to determine if the staining represents apoptosis or autophagy after the samples were quantified.
Using the simple Photoshop method demonstrated earlier, results confirmed that increasing concentrations of hydrogen peroxide resulted in a higher level of lys tracker staining Once mastered, this staining procedure can be done in less than two hours if it is performed properly Following this procedure. Other methods like NX in five or lc three immuno staining can be performed in order to confirm the type of cell death. Don't forget that currently Trecker green only works on live cells, so Reca red is your best option for samples that require fixation.
