Anh:  We usually culture for 10 passages for experiments after expanded from ATCC stock.  As you don't know how many passages your student put into LN tank many years ago, I'd be careful in using the cells at all unless you have some assay to confirm that the cells still behave as typical melanocytes.  As to the spin speed, you only need to get cells to the tube bottom and can be collected.  So, 500 g for 5 min should be enough.  Good luck.

 Ke-Wei Zhao: Ah thank you very much. But can you suggest me some kind of assay to test for melanocyte characteristics? When look under the microscope, they all look fine, healthy and everything. But how do you really know? 

Anh: As melanocytes contain melanosomes, a unique lysosomal vesicle containing melanin, I think you can look for that or stain it with an anti-melanosomal antibody, HMB45.  That should be unique to be sure that the cells are not ordinary fibroblasts or keratinocytes.

Well the melanosome is ok, but what about the actual characteristic of the cell line. As in how would you know if the cells still behave the way it should be. Because sometimes when you look under the microscope, the phenotype and everything looks fine, but the internal behavior may have changed. But I guess there isnt any quick available test or some sort of feature to quickly recognise that, is it? 

Hi,

I just found your question.  There is a lot of information around, about how long mouse cells will grow.  Actually primary normal mouse melanocytes grown with serum, TPA and CT will grow only for about 10 population doublings (maybe 3-4 passages depending on your protocol) before senescing.  But if someone has grown enough cells to freeze down, then they have very likely immortalized, or were taken from a Cdkn2a-null mouse so they are naturally immortal.  In that case they will grow forever.  But they will gradually get less differentiated as you passage them for longer or freeze them more times. (Highly pigmented cells tend to die upon freezing, unless you include a tyrosinase inhibitor like PTU in the freezing and thawing medium.)  For our melan-a melanocyte line (for example) we recommend not going above passage 30 (counting from first explantation) if you want to avoid dedifferentiated or transformed cells. 

The easiest "assay" for all the cells being melanocytes is cytoplasmic melanin.  If you set up your microscope for perfect bright-field (condenser focussed, condenser iris wide open, turn down lamp if too bright), then melanocytes are still visible by their pigment, whereas other cells disappear.  The cell shape is also very characteristic.  You could check our papers for that (starting in 1987 - mostly available on ReseachGate).

Hello Professor Bennett,

Thank you so much for such an informative comment. My cells are immortalised, indeed. They are Ink4a/Arf-/-. 

I don't think we ever added Tyrosinase inhibitor. Previous students preserve cells in liquid nitrogen and when I took them out and regrow them again, they look fine, so I assume at least I can still recover them back. I asked people around and they have told me that the cells can even survive at -80 in freezing media for up to 2 years, but we would still gonna transfer them to liquid nitrogen anyway. 

I could still observe black pigmentation of these cells under the microscope so I assume they are still melanocytes. But the method you suggest will be very handy for me indeed. 

So thank you very much for the informative comment. 

Besides trying to revive and passage INK/Arf-/- frozen cells, you can also isolate primary melanocyts from the homozygous neonates and start fresh. You may work with what you have and get some data but questions will be asked about the passage level you used for doing studies with the cell line. I would suggest keepng an eye on their doubling potential and cellular morphology (proliferative vs differentiated) with every passage since these characteristics change with long passages.

Hi, glad to see these comments.

I am in the other side of the Atlantic, but desperately looking for immortalized melanocytes...

Does anyone have a protocol to isolate them from mouse neonates? or even older?

Maria

We have dozens of immortal mouse melanocyte lines, mostly published over the years, or see our Cell Bank site.

https://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm

We also have protocols (also published) if you want to start again yourself. Unless the mice are Cdkn2a (p16-Arf) null, it will take about 6-8 months to get them spontaneously immortal and grow up the immortal cells. (If Cdkn2a null, it will take maybe 4-6 weeks just to grow them up, as the whole culture will be already immortal.)

Hi,

I agree to the response of Prof. Bennet. In addition, you can also perform DOPA staining to confirm the melanocyte characteristics.