Jun 21, 2023
Single cell dissociation of healthy paediatric skin
- Emily Stephenson1,2,
- Chloe Admane1,2,
- Keval Sidhpura1,2
- 1Newcastle University;
- 2Wellcome Sanger Institute
Protocol Citation: Emily Stephenson, Chloe Admane, Keval Sidhpura 2023. Single cell dissociation of healthy paediatric skin . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd6kwlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2023
Last Modified: June 21, 2023
Protocol Integer ID: 83638
Funders Acknowledgement:
CZI Pediatric Networks for the Human Cell Atlas
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.
Guidelines
This protocol includes an overnight incubation step.
Materials
Petri dish
Scalpel
Forceps
100 micron fiilters
RPMI
RF-10 (RPMI plus 10%FCS, 1% Pen-strep and 1% L-glut)
48-well v bottom plate
PBS
Dispase (Roche)
Collagenase Type IV (Worthington)
50ml Falcon Tubes
Flow Buffer (PBS 2% FCS and 2mM EDTA)
Safety warnings
This protocol uses sharp objects.
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.
Day 1 - Begin protocol late afternoon
Day 1 - Begin protocol late afternoon
Record sample meta data and assess size of sampleSample
10m
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
5m
Cut off lower dermis and fat and place in well of 48-well plate with 1ml RPMI
2m
Place epidermis and upper dermis sample in a 48 well V-bottom plate with 1ml RPMI and 20µL Dispase
5m
Add parafilm to plate and leave in 4°C fridge overnight
1m
Day 2 - Begin protocol early in the morning
Day 2 - Begin protocol early in the morning
4h 8m
4h 8m
Take plate out of fridge and empty epidermis/upper dermis onto new petri dish, remove collagenase type IV from -80 freezer
2m
Using forceps, separate epidermis and upper dermis and place each separately into new wells of the 48-well plate
10m
Place 1ml RPMI in each well of plate containing epidermis and upper dermis
5m
Add collagenase type IV 1:100 (10µL) to each of the tissues (epidermis, upper dermis and lower dermis)
5m
Place in incubator and incubate at 37°C for 3 hours
3h
Remove from incubator and, using 1ml pipette, pipette each tissue up and down to ensure the tissue has dissociated
10m
Pipette through 100micron filter into separate 50ml Falcon tubes
5m
Wash out each well an additional 3 times with 1ml RF-10 and pass through filter
2m
Wash filter with 25ml RF-10 and adjust so each Falcon tube has the same volume
2m
Centrifuge at 500g for 5 mins at 4deg (9acc/dec)500 x g, 4°C
5m
Pour off supernatant
2m
Resuspend pellet with 1ml of Flow Buffer
5m
Take 10µL for cell count and count using Trypan Blue and C-Chip Haemocytometer, record number of cells isolated for each tissue
15m
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