Single cell dissociation of healthy paediatric skin
Emily Stephenson, Keval Sidhpura, Chloe Admane
Disclaimer
This protocol has been tested on a variety of skin sites such as lip and trunk, however not all body sites has been tested. This protocol has also only been used for healthy, non-diseased skin.
Abstract
This protocol outlines the method for the enzymatic dissociation of healthy paediatric skin >3mm into a single cell suspension.
Before start
Do not forget to record the metadata for this sample. Before starting, clean MSC Class II with 70% ethanol and make up virkon. Ensure you have all materials needed to carry out the protocol.
Steps
Day 1 - Begin protocol late afternoon
Record sample meta data and assess size of sampleSample
If sample is <3mmx3mm freeze and embed sample in OCT (see other protocol), if sample is >3mmx3mm continue with protocol
Empty sample onto petri dish and wash sample with PBS
Cut off lower dermis and fat and place in well of 48-well plate with 1ml RPMI
Place epidermis and upper dermis sample in a 48 well V-bottom plate with 1ml RPMI and 20µL Dispase
Add parafilm to plate and leave in 4°C fridge overnight
Day 2 - Begin protocol early in the morning
Take plate out of fridge and empty epidermis/upper dermis onto new petri dish, remove collagenase type IV from -80 freezer
Using forceps, separate epidermis and upper dermis and place each separately into new wells of the 48-well plate
Place 1ml RPMI in each well of plate containing epidermis and upper dermis
Add collagenase type IV 1:100 (10µL) to each of the tissues (epidermis, upper dermis and lower dermis)
Place in incubator and incubate at 37°C for 3 hours
Remove from incubator and, using 1ml pipette, pipette each tissue up and down to ensure the tissue has dissociated
Pipette through 100micron filter into separate 50ml Falcon tubes
Wash out each well an additional 3 times with 1ml RF-10 and pass through filter
Wash filter with 25ml RF-10 and adjust so each Falcon tube has the same volume
Centrifuge at 500g for 5 mins at 4deg (9acc/dec)500x g,4°C,0h 0m 0s
Pour off supernatant
Resuspend pellet with 1ml of Flow Buffer
Take 10µL for cell count and count using Trypan Blue and C-Chip Haemocytometer, record number of cells isolated for each tissue
If sorting for single cell RNA seq, continue with antibody staining and FACS protocol or if freezing down cells, continue with viable cell freezing protocol
