TITLE: Single cell dissociation of healthy paediatric skin 
AUTHORS: Emily Stephenson, Chloe Admane, Keval Sidhpura

[DESCRIPTION]
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.

[GUIDELINES]
This protocol includes an overnight incubation step.

[STEPS]
SECTION: Day 1 - Begin protocol late afternoon
2. Empty sample onto petri dish and wash sample with PBS
SECTION: Day 1 - Begin protocol late afternoon
3. Cut off lower dermis and fat and place in well of 48-well plate with 1ml RPMI
SECTION: Day 1 - Begin protocol late afternoon
4. Place epidermis and upper dermis sample in a 48 well V-bottom plate with 1ml RPMI and 20µL Dispase
SECTION: Day 2 - Begin protocol early in the morning
6. Take plate out of fridge and empty epidermis/upper dermis onto new petri dish, remove collagenase type IV from -80 freezer
SECTION: Day 2 - Begin protocol early in the morning
7. Using forceps, separate epidermis and upper dermis and place each separately into new wells of the 48-well plate
SECTION: Day 2 - Begin protocol early in the morning
8. Place 1ml RPMI in each well of plate containing epidermis and upper dermis
SECTION: Day 2 - Begin protocol early in the morning
9. Add collagenase type IV  1:100 (10µL) to each of the tissues (epidermis, upper dermis and lower dermis)
SECTION: Day 2 - Begin protocol early in the morning
10. Place in incubator and incubate at 37°C for 3 hours
SECTION: Day 2 - Begin protocol early in the morning
11. Remove from incubator and, using 1ml pipette, pipette each tissue up and down to ensure the tissue has dissociated
SECTION: Day 2 - Begin protocol early in the morning
12. Pipette through 100micron filter into separate 50ml Falcon tubes
SECTION: Day 2 - Begin protocol early in the morning
13. Wash out each well an additional 3 times with 1ml RF-10 and pass through filter 
SECTION: Day 2 - Begin protocol early in the morning
14. Wash filter with 25ml RF-10 and adjust so each Falcon tube has the same volume
SECTION: Day 2 - Begin protocol early in the morning
15. Centrifuge at 500g for 5 mins at 4deg (9acc/dec)500 x g, 4 °C 

SECTION: Day 2 - Begin protocol early in the morning
16. Pour off supernatant
SECTION: Day 2 - Begin protocol early in the morning
17. Resuspend pellet with 1ml of Flow Buffer
SECTION: Day 2 - Begin protocol early in the morning
18. Take 10µL for cell count and count using Trypan Blue and C-Chip Haemocytometer, record number of cells isolated for each tissue
SECTION: Day 1 - Begin protocol late afternoon
5. Add parafilm to plate and leave in 4°C fridge overnight
SECTION: Day 1 - Begin protocol late afternoon
1. Record sample meta data and assess size of sample  

SECTION: Day 1 - Begin protocol late afternoon
1.1. If sample is <3mmx3mm freeze and embed sample in OCT (see other protocol), if sample is >3mmx3mm continue with protocol
SECTION: Day 2 - Begin protocol early in the morning
18.1. 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