Shreffler:DC Isolation from skin: Difference between revisions
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# 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. | # 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. | ||
# Mount the strips on a dissection board and cut split thickness skin grafts use the Webster. | # Mount the strips on a dissection board and cut split thickness skin grafts use the Webster. | ||
#: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. | |||
# Incubate with dispase 1U/ml 60-120 minutes 37 in RPMI or X-Vivo - skin area should not exceed the area of the dish | # Incubate with dispase 1U/ml 60-120 minutes 37 in RPMI or X-Vivo - skin area should not exceed the area of the dish | ||
# 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. | # 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. | ||
# For migration: | # For migration: | ||
# | ## put the dermal sheet into X-vivo, no serum of GFs required and leave 48-72h -this yields the classic maturing CD14+ CD1a+ DC with most of the dermal lymphocytes | ||
# For collagenase digestion: | # For collagenase digestion: | ||
# | ## put the sheets into RPMI 10% FBS with 0.8mg/ml collagenase (worthington IV) for 8-12h. We usually do this step overnight. | ||
##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. | |||
##Filter the remnants out. 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== | ==Discussion== |
Revision as of 07:39, 12 December 2009
Overview
Materials
Equipment
- Goulian Skin Graft Knife
- Dissection Board
- Scalpel
- Flat forceps
- Low binding culture dishes
Reagents
- X-vivo
- Dispase
- Collagenase
Procedure
- 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.
- Mount the strips on a dissection board and cut split thickness skin grafts use the Webster.
- 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.
- Incubate with dispase 1U/ml 60-120 minutes 37 in RPMI or X-Vivo - skin area should not exceed the area of the dish
- 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.
- For migration:
- put the dermal sheet into X-vivo, no serum of GFs required and leave 48-72h -this yields the classic maturing CD14+ CD1a+ DC with most of the dermal lymphocytes
- For collagenase digestion:
- put the sheets into RPMI 10% FBS with 0.8mg/ml collagenase (worthington IV) for 8-12h. We usually do this step overnight.
- 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.
- Filter the remnants out. 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
References
-
pmid= 19171766
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare