How to extract the little secret of melanocytes

How to extract the little secret of melanocytes

Experimental dogs always have to do the experiment, this is not, just do the week cells, melanocytes and climb the mountains and wade in the rush to come over, pick up surgical scissors, forceps and other experimental small helper began to operate it!

Tissue sample cleaning process

a) Take the skin with pores, 1cm×1cm in size, be sure to have hair follicles, soak in alcohol for 1 minute, rinse twice with pre-cooled 49%PBS+49%MEM+2%PSN, and then remove the subcutaneous connective tissue;

b) Use ophthalmic scissors to cut the skin into 0.2cm×0.5cm skin blocks, and put them into 20mL of Dispase II digestion solution, be sure to immerse them, and digest at 4℃ overnight for 14-16h;

c) On the second day, when the epidermis and dermis showed signs of separation in the tissue incision, the skin was placed in a dry petri dish and peeled off with eye tweezers. If the epidermis could not be peeled off easily, the skin was placed in DispaseII enzyme again and incubated at 37℃ for 15-30min;

d) The removed epidermis was collected in a 35mm dry petri dish, and 1ml 0.25% pancreatic enzyme was added for digestion at 37℃ for 8min;

e) Add 2mL serum to terminate digestion, blow it several times, filter it with a 200-mesh cell filter, and then transfer it to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min;

f) Adding 2mL of melanin to complete medium suspension, placed in 35mm petri dish;

g) After 1h-2h culture, the adhesion of melanocytes to the wall was observed by microscope, and the suspended cells were removed by fluid change, which were keratinocytes, and then the medium was changed every 2 days

DAY5: Melanocyte and HMSA immunofluorescence staining

Isn't it simple, not only to extract melanin but also to take keratinocytes?

There's one more secret I want to tell you

Use the following artifacts to easily get the cells you want

MM melanocyte medium:

1. Low serum component, serum content is 1%

2. Add small molecule compounds that block keratinocytes and proteins that promote melanocyte growth to let melanocytes grow freely

3. The medium does not contain TPA carcinogenic small molecules to avoid the carcinogenesis of black cells in the process of expansion

KGM keratinocyte medium

1. Set, easy to use;

2. ITS+E was used to replace the serum-free components of BPE to reduce the interference of animal-derived protein;

3. After testing of various keratinized cell lines and primary cells, the components were stable and the cell growth was good