I culture primary melanocytes and Keratinocytes from neonatal foreskin. I usually get pretty good cultures and for some experiments i wanna have pure keratinocytes (no melanocytes at all).
Can anyone suggest me how to get rid of melanocytes (if any) from keratinocyte culture and to make sure that what i have is pure Keratinocytes.
Thanx....
There are papers to obtain pure melanocytes (as J Invest Dermatol. 1983 Aug;81(2):179-80. A procedure for the isolation of primary cultures of melanocytes from newborn and adult human skin. Tsuji T, Karasek M.), but as far as I know, there is no similar methods for keeping pure keratinocytes. However, in my experience, keratinocytes use to growth much quicker than melanocytes, and when they are confluent, they form a sheet of keratinocyte that it can be detached with very few (if any) melanocytes. Hit the flask carefully to get a differential cell detaching. After few passages, you should have pure keratinocytes.
I hope this will help you.
Dear Dr. Solano
Thanx for ur suggestions.
Actually i culture melanocytes and keratinocytes from epidermis of neonatal foreskin.The culture so obtained is a mixed culture and i perform differential trypsinization to separate melanocytes from keratinocytes. However, few melanocytes remain with keratinocytes.
I was just wondering that ,is there a method where melanocytes can be killed in keratinocyte culture(like G418 treatment is given to get rid of fibroblasts in melanocyte culture).
Anyways i will go through the papers you suggested.
thanks a lotzz.
Try sorting melanocytes using CD117 (C-KIT) antibody either using Miltenyi magentic bead system for FACS sorting. This will eliminate melanocytes from your culture and leaving you with mostly keratinocytes . CD117 specifically binds to melanocyte receptors.
Hi Utpreksha,
Are you still facing this problem ? If yes - what is the ratio of the M:K in your culture? If it low then the melanocytes eventually die in subsequent splitting as the keratinocyte media wont aid the growth of melanocyte media for long.
Hi Arisa,
The number of melanocytes is actually very less and yes their number go down in subsequent passages.
Thanku.
which type of culture system you are using for keratinocyte culture. Feeder or feeder less????
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