The overall goal of this protocol is to use the small molecule compounds PD0325901 and Vitamin C to maintain a hypomethylated and pluripotent state in mouse embryonic stem cells. This method can help answer key questions in the field of FBS cultured mouse embryonic stem cells, such as heterogenerity in morphology and the dehypermethylation. So the main advantage of this technique is that the mouse embryonic stem cells are able to sustain excellent morphology and the hypomethylated and the undifferentiated state.
After preparing plates and buffers, according to the text protocol, prewarm the mouse ES cells, culture medium, trypsin, and PBS in a water bath at 37 degrees Celsius. To passage the cells, aspirate the medium from the dish and use two milliliters of PBS to rinse the cells two times. Then remove the PBS and add 0.3 milliliters of trypson EDTA to the cells and quickly tilt the dish to cover the cells in the solution.
Immediately remove the trypsin and incubate the plate at 37 degrees Celsius for one minute to detach the cells. Add two milliliters of pre-warmed serum medium to inactivate the trypsin and use a five milliliter pipet to re-suspend the cell colonies 10 times into a single cell suspension. Plate 150 microliters of the cell solution into a new six centimeter, gelatin-coated dish and supplement with three milliliters of freshly made VCPD0325901 medium.
Culture the cells at 37 degrees Celsius and five percent CO2. Due to the instability of Vc, every 24 hours while incubating, remove the old culture medium and add three milliliters of fresh Vcpd0325901 medium. After reaching 70 to 80 percent confluency, passage the cells and continue to culture them as just demonstrated.
To carry out DNA extraction, collect the cells with trypsin as demonstrated earlier and use a genomic DNA purification kit following the manufacturers instructions. Add 100 microliters of ultra pure water to the tube of DNA and dissolve the DNA by pipetting approximately 15 times. Then, use a spectrophotometer to measure the Od260 to 280 ratio to determine the concentration and quality of DNA.
The DNA concentration should be about 500 nonograms per microliter. Next, digest five micrograms of DNA by combining it with five microliters of 100 millimolar tris hydrochloride PH7.6, two units of calf intestinal phosphatase, one unit of Dnase one, and 0.005 units of snake venom, phosphodyesterase one. Then, use ultra pure water to bring up the volume to 50 microliters.
Incubate the reaction at 37 degrees Celsius over night. The following morning, collect the sample by centrifugation at 1, 000xg at room temperature for one minute. Then transfer the solution into ultra filtration tubes and spin the samples at 13, 000xg and 4 degrees Celsius for 30 minutes to remove the digestion enzymes.
To prepare the samples for 5HMC analysis, transfer 36 microliters of the filtrate to a new one milliliter tube and add four microliters of D35HMC for a final concentration of three nanomolar. For 5MC analysis, pipet 196 microliters of ultra pure water into a new centrifuge tube and add four microliters of filtrate due to the high density of 5MC in genomic DNA. Transfer 36 microliters of the diluted 5MC solution to a new centrifuge tube and add four microliters of D35MC for a final concentration of 50 nanomolar.
Use D35HMC and D35MC as internal standards to calibrate 5HMC and 5MC respectively. After setting up the parameters to analyze 5MC and 5HMC in the UHPLCMS software, according to the text protocol, establish the parameters for QQQMSMS by clicking MS QQQ. For stop time, choose No limit/As Pump.
For ion source, choose ESI. For time filtering, check Peak width and enter 0.07 minutes. To set up the time segments, for start time, choose zero.
To set scan mode in MRM to analyze the allusion from the column, for scan type, choose MRM. For Div Value, choose To MS.For Delta EMV plus, enter 200 and for Delta EMV minus, enter zero. Build the parameters for monitoring the transitions by first clicking MSQQQ one acquisition.
To set scan segments, for Dwell, enter 90. To set the fragment voltages for the Fragmentor, enter 90. For Collision Energy, enter five.
For the Cell Accelerator Voltage, enter four. To set the positive ion mode, for Polarity, choose positive. To carry out UHPLC separation of mononucleosides, prepare solutions a and b for the allusion of 5MC and cytosine by mixing 500 milliliters of ultra pure water with 500 microliters of formic acid for solution a.
Then measure 500 milliliters of 100 percent methanol and solution b. Mix solution a and solution b at a 95 to five volume to volume ratio as the mobile phase to elute 5MC and C.Then set the flow rate to 0.25 milliliters per minute. Separate 5HMC using an optimized gradient allusion.
Then set the flow rate at 0.25 milliliters per minute. Monitor the transitions for 5MC, D35MC, DC, 5HMC, and D35HMC. Set the capillary voltages at 3, 500 volts.
Then set the injection volume to 5 microliters and analyze each sample three times. Employ the corresponding standard curves to evaluate the amount of 5MC and 5HMC according to the text protocol. As seen in this analysis of genomic DNA in mouse ES cells by UHPLC MSMS, VcPD0325901 treatment resulted in a more rapid decline in DNA methylation and reached steady five MC levels after five days, while Vc 2i treatment resulted in a comparable level at day 11.
This graph shows that Vc containing treatment dramatically increased 5HMC frequency after one day and thereafter, 5HMC levels gradually declined due to a progressive reduction in 5MC substrate. Western blot analysis showed that Prdm14 was significantly elevated, while Dnmt3b and its co-factor, Dnmt3l, were considerably down regulated when cells retreated with PD0325901, indicating the action of PD0325901 in erasing 5MC. In this alkaline phosphatase assay, mouse ES cells were all stained purple with a ball-like morphology when cultivated for 26 days in VcPD032590, indicating an undifferentiated state.
In contrast, cells grown in FBS containing medium alone appeared light in color with partial outspreading indicating partial differentiation. Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it's important to remember that FBS need to be pre-screened.
Also, avoid over-pipetting cells through the passage and changing the VcPDO325901 culture medium daily. After this development, this technique paves the way for researchers in the field of culturing mouse ES cells in vitro to explore the development and processes of embryos in vitro and the generated cells of medical relevance for regenerative medicine. After watching this video, you should have a greater understanding of how to maintain growth morphology and hypermethylated and plural potent state for mouse ES cells by using two small molecular components, the MEK inhibitor, PD0325901.
Don't forget that working with Trisol, DMS or PMS and the DTT can be extremely hazardous and the precautions such as wearing gloves, a mask, and the goggles should always be taken while performing this procedure.
