Shreffler:DC Isolation from skin

Overview
This protocol generously shared by Matthew Collin. Published in J Exp Med 2009. Haniffa

Equipment

 * Goulian Skin Graft Knife
 * Dissection Board
 * Scalpel
 * Flat forceps
 * Low binding culture dishes
 * 70 μm mesh filters

Reagents

 * X-vivo
 * Dispase
 * Collagenase

Procedure

 * 1) Cut the skin into strips about 1cm wide with a scalpel. Also remove all the subcut tissue if this has not already been done by the surgeon.
 * 2) Mount the strips on a dissection board and cut split thickness skin grafts using the skin knife.
 * This requires a little practice: usually pin one end and pull tight with a large flat forceps then gently cut with lots of side to side movement - don't expect the knife to travel too quickly. Works best on strips about 10-15cm long. Cut skin should be almost translucent.
 * 1) Incubate with dispase 1U/ml 60-120 minutes 37  in RPMI or X-Vivo - skin area should not exceed the area of the dish
 * 2) Peel into epidermis and dermis with forceps - if nicely done the sheets should just give with a little tension, not fall apart or not break with too much tension.
 * 3) For migration:
 * 4) put the dermal sheet into X-vivo, no serum or GFs required and leave 48-72h.
 * this yields the classic maturing CD14+ CD1a+ DC with most of the dermal lymphocytes
 * 1) For collagenase digestion:
 * 2) Put the sheets into RPMI 10% FBS with 0.8mg/ml collagenase (worthington IV) for 8-12h.
 * We usually do this step overnight.
 * 1) Resuspend with a 10ml pipette rather than shearing with a needle (viability drops if you do this).
 * Pipetting should swirl the tissue into a suspension quite easily if the digestion is good. Main cause of failure to do this is sheets too thick or culture plate has too much tissue in it.
 * 1) Filter the remnants out with 70 μm mesh filter.
 * This yields everything and then you can sort relatively 'fresh' DC, macrophages, Mast cells etc. You can also use LPS-free liberase preps if you want the least perturbed DC, but the enzyme is more expensive.

Discussion
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Contact

 * Who has experience with this protocol?
 * Email Wayne Shreffler through OpenWetWare