July 14, 2009 - 1:51am
Eyal Ozeri
Joined: Jul 14 2009
hello ,
I am trying to isolate Langerhans cells from skin and ears of mice, but haven't found any success.
I have looked at many protocols from various papers and they all say the same: incubate the skin with 0.5-0.25% trypsin with PBS over night .
if someone has any tips, i will be more then grateful.
Thanks!
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The classical approach to isolate Langerhans cells (LC) from skin is indeed trypsinization of the epidermis and subsequent enrichment for DC cells. Did you incubate the tissue at 37ºC and did you try higher trypsin concentrations. Schuler and Steinman (1985) use a modified protocol to isolate succesfully LC cells from mouse ear skin by placing ear halves in HBSS and incubating with 2.5% trypsin for 30 min at 37°C (i.e., using 10-fold higher trypsin concentration than you used).
An alternative method to the trypsin-based approach is to essentially float mouse ear halves in standard culture medium so that DC cells spontaneously emigrate. This method was first described by Larsen et al (1990) and later modified by Ortner et al (1996). In the original procedure skin explants were cultured for a period of 3 days in the same culture well, whereas Ortner et al transferred skin explants to fresh wells each day, which resulted in higher absolute numbers and higher percentages of enrichment. The skin culture technique is very similar to the enzymatic approach in terms of absolute cell numbers and enrichment. However, preparing ear explants for culture is much less of an effort than preparing epidermal cell suspensions and enriching for Langerhans cells. Furthermore, trypsin is not used in the skin culture approach. Please refer to the two papers for details of the described protocols: Schuler and Steinman (J Exp Med. (1985) 161(3) :526-46) and Ortner et al (J. Immunol. Methods. (1996) 193(1) :71-9).