Hello Sunyna Saun

The key factors for achieving uniform differentiation in HaCaT cells include a combination of cell density, calcium concentration in the medium, and time in culture.

When HaCaT cells reach around 80% confluency, it is a good starting point for differentiation experiments. This density allows for cell-to-cell contact, which is important for keratinocyte differentiation.

The extracellular calcium concentration is a key regulator of keratinocyte differentiation. You may culture the cells in low-calcium (0.03 mM) DMEM with 10% chelexed FBS (FBS that has been treated with a chelating agent, typically Chelex, to remove excess metal ions), and 2mM L-Glutamine to keep them in a proliferative, undifferentiated state. Once the desired cell density is reached (80%), switch the culture medium to a higher calcium concentration (e.g., 2.8 mM) to initiate differentiation by promoting cell-cell adhesion and activating differentiation-related genes like keratin 1 and involucrin.

If you desire, you may perform a time course study where in you may harvest samples at regular intervals (e.g., every 24 hours for 3-5 days) after the calcium switch to observe the progression of differentiation. You may confirm differentiation by checking the expression levels of differentiation markers such as keratin 1, keratin 10, and involucrin. Alpha-tubulin may be used as a housekeeping marker for normalization.

So, if you carefully control the above factors, you can improve the uniformity of differentiation in HaCaT cells.

Best,