Hi, I'm Sarah NAU at the STEM Gen Laboratory in Boston. Today we'll show you a procedure for maintaining self-renewal of mouse embryonic stem cells in the presence of the small molecule as C one mouse. Embryonic stem cells are normally cultured in the presence of leukemia inhibitory factor to maintain self-renewal.
L however, is expensive. So here we'll provide an economical alternative, the small molecule SC one. In the last part of the video, we'll be showing you a comparison of pluripotency marker expression between cells cultured in L and those cultured in SC one.
So let's get started. One day before culturing the embryonic stem cells thaw a vial of irradiated feeder cells. Plate the feeder cells at a density of 10, 000 cells per well.
In a 12 well plate incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. The next day plate. The ES cells on the MEF feeder layer seed the cells at a density of 50, 000 cells per well on the prepared feeder plates In growth media supplemented with SC one at the desired concentration we used 100 nano molar, 300 nano molar, and one micromolar.
As a control. We add a well that was supplemented with 1000 micrograms per milliliter of LIF. Now incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide.
The next day refresh the media for continuous culturing. Refresh the media every 48 hours passage the cells, one per 10 every three to four days to maintain a similar density to the initial seating density. After five passages, we can now check for the expression of typical pluripotency markers using immunochemistry to begin immunochemistry of stem cells.
Start by washing the cells gently three times with PBS. Fix the cells with 500 microliters of 4%para formaldehyde for 20 minutes at room temperature. After washing the cells in PBS, three more times, block non-specific binding with 500 microliters of blocking buffer for one hour at room temperature.
Next, add 250 microliters of the primary antibody to the cells and allow them to incubate at four degree Celsius overnight. In this experiment, we are incubating with nano SOX two SSEA one, and OCT four antibodies. After another set of washes incubate the cells with 250 microliters of the fluorescent dye conjugated secondary antibody for two hours at room temperature, keeping them away from light, wash the cells a final time adding DAPI at a final concentration of one microgram per milliliter to the last wash.
Then incubate the cells for five minutes to stain the nuclei. Finally, analyze the cells under fluorescent microscope. Shown here are brightfield images of mouse ES cells grown on feeder cells in the presence of either LIF or SC one to sustain self-renew at an undifferentiated state.
Here, mouse ES cells are shown, which have been maintained for five passages. The cells were fixed and examined by immuno cyto chemistry for SSEA one OCT four and nano and SOX two. Marker expression for cells maintained in C one was similar to cells maintained in LIF.
We've just shown you how to use the small molecule SC one to maintain self-renewal in mouse embryonic stem cells. When doing this procedure, it is important to remember that SC one should be titrated to determine the optimal concentration before being used on an untested cell line. So that's it.
Thanks for watching and good luck with your experiments.
