Shreffler:DC Isolation from skin: Difference between revisions
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==Overview== | ==Overview== | ||
This protocol generously shared by Matthew Collin. Published in J Exp Med 2009.<cite>Haniffa</cite> | |||
==Materials== | ==Materials== | ||
===Equipment=== | ===Equipment=== | ||
*Goulian Skin Graft Knife | * Goulian Skin Graft Knife | ||
*Dissection Board | * Dissection Board | ||
*Scalpel | * Scalpel | ||
*Flat forceps | * Flat forceps | ||
*Low binding culture dishes | * Low binding culture dishes | ||
* 70 μm mesh filters | |||
===Reagents=== | ===Reagents=== | ||
*X-vivo | * X-vivo | ||
*Dispase | * Dispase | ||
*Collagenase | * Collagenase | ||
==Procedure== | ==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. | # 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 | # 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. | #: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 | ## 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 | ##:this yields the classic maturing CD14+ CD1a+ DC with most of the dermal lymphocytes | ||
# For collagenase digestion: | # For collagenase digestion: | ||
Latest revision as of 17:25, 1 February 2010
Overview
This protocol generously shared by Matthew Collin. Published in J Exp Med 2009.[1]
Materials
Equipment
- Goulian Skin Graft Knife
- Dissection Board
- Scalpel
- Flat forceps
- Low binding culture dishes
- 70 μm mesh filters
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 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.
- 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 or GFs required and leave 48-72h.
- this yields the classic maturing CD14+ CD1a+ DC with most of the dermal lymphocytes
- put the dermal sheet into X-vivo, no serum or GFs required and leave 48-72h.
- 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 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.
- Put the sheets into RPMI 10% FBS with 0.8mg/ml collagenase (worthington IV) for 8-12h.
Discussion
References
- Haniffa M, Ginhoux F, Wang XN, Bigley V, Abel M, Dimmick I, Bullock S, Grisotto M, Booth T, Taub P, Hilkens C, Merad M, and Collin M. Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation. J Exp Med. 2009 Feb 16;206(2):371-85. DOI:10.1084/jem.20081633 |
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare
