Isolation of Skin Cell Protocol
The following protocol outlines the digestion of skin cells from tissue samples stored in CryoStore (CS) 10 media. All consumables and recipes for necessary media and solution are listed below. This protocol has an optional step for staining cells for CITEseq experiments
Consumables
Item | Manufacturer or Catalog No. | Storage Location | Notes |
Collagenase P | Merk 11213857001 | 4°C Fridge | 100mg lyophilized. See below for resuspension instructions |
DNase I | Cell Center | -20°C Freezer | |
UltraPure 0.5M EDTA | Invitrogen 15575020 | Room Temperature (Cabinet above Thermal Cyclers) | |
UltraPure BSA | Invitrogen AM2618 50 mg/mL | -20°C Freezer | |
PBS | Cell Center | Room Temperature (Cabinet above Thermal Cyclers) | |
Molecular Grade Water | Cell Center | Room Temperature (Cabinet above Thermal Cyclers) | |
FBS | Cell Center | -20°C Freezer or -20°C Freezer above 4°C Fridge | Check for aliquots before opening new bottle |
RPMI 1640 | Cell Center | 4°C Fridge | |
Penicillin/Streptomycin | Cell Center | -20°C Freezer or -20°C Freezer above 4°C Fridge | Check for aliquots before opening new bottle |
L-Glutamine | Cell Center | -20°C Freezer or -20°C Freezer above 4°C Fridge | Check for aliquots before opening new bottle |
Other Materials
Item | Catolog No. | Storage Location | Notes |
Sterile Scalpel or Scissors | Drawer 16 | ||
60mm x 15mm Sterile Petri Dish | Below NextSeq 2000 | ||
Vacuum Filter Bottle | Below NextSeq | ||
6 Well Plate | Below NextSeq 2000 | Only if doing multiple samples | |
70 µM Cell Strainer | |||
40 µM FloMi Cell Filter | |||
Sterile Needle | |||
1 mL Syrine | |||
37°C Water Bath | For this we use a Sous Vide set to 37°C and a plastic bucket |
1. Make all necessary Solutions
The following solutions will be needed to conduct this protocol. For best results make fresh the day of the cell digestion.
R10 Solution
- Set up 37°C Water Bath to thaw any reagents frozen at -20°C
- Gather the following:
- RPMI 1640
- 50 mL FBS aliquot
- 5 mL Pen/Strep aliquot
- 5 mL L-Glutamine aliquot
- Thaw frozen reagents for 10 min or until completely thawed.
- Open the package of the vacuum filter bottle under the biosafety hood! Connect a vacuum filter bottle to a vacuum line in a biosafety hood. (The Scott Lab can provide this space).
- Combine all reagents in the top of a Vacuum Filter bottle.
- Slowly turn on the vacuum line to begin filtering.
- Once all liquid has been filtered, carefully remove the top of the bottle and discard the top portion and screw on the included cap.
- Invert to mix R10 solution.
- Store at 4°C
R10 + Collagenase P Solution
- Gather the following:
- R10 Solution
- 100 mg Collagenase P
- The Collagenase P will ship as 100 mg, lyophilized in a small class container with a rubber stopper. Do not remove the rubber stopper!
- Using an aliquot of R10 Solution, aspirate 1 mL of R10 using a sterile needle and syringe.
- Puncture the syringe through the rubber stopper and dispense 1 mL of R10 solution into the glass container
- Invert to mix until the collagensase is completely dissolved.
- Using the same needle, aliquot 100 uL (0.1 mL) if conducting the cell digest the same day. Otherwise store at 4°C.
10mM EDTA
- Gather the following:
- 0.5M EDTA
- 1x PBS
- Combine 1 mL of 0.5M EDTA in 49 mL of 1x PBS in a new 50 mL Conical
- Store at 4°C.
10mM EDTA
- Gather the following:
- BSA
- 1x PBS
- Combine 2µL of BSA in 25 mL of 1x PBS in a new 50 mL Conical
- Store at 4°C.
2. Prepare and Digest Sample
Thaw and Prepare Tissue
- If water bath is not set up, prepare a 37°C water bath
- Acquire tissue sample(s) from -80°C Freezer.
- Incubate sample for 10 min at 37°C.
- Move sample to biosafety hood that contains the following:
- 4 mL of 1x PBS
- Sterile Petri dish
- Scalpel/Scissors
- R10 solution
- R10 + Collagenase P aliquot
- Wash sample in tube with 1x PBS for a total of 3 washes.
- Remove CS 10 solution
- Add 1 mL of 1xPBS to tube containing tissue sample
- Remove 1 mL of supernatant
- Repeat steps b-c for a total of 3 washes
- Transfer tissue sample to the Petri dish. Use a clean P1000 pipette tip if necessary to help remove the sample from the tube
- Cut the skin into small pieces using the scalpel and scissors. Tissue will be very tough!
- Add 40 µL of 100mg/mL Collagenase P in R10 to 4 mL of R10 solution. Add the entire mixture to the petri dish. Gently swirl the petri dish.
- Alternatively, add 4 mL of R10 to the petri dish. Next add 40µL of Collagenase in R10 to the petri dish. Gently swirl the petri dish
- Incubate the petri dish in an incubate at 37°C for four hours. (The Scott Lab has one directly next to the biosafety hood in their tissue culture room).
- At the end of the 4 hour incubation, add 4 uL of 100 mg/mL DNaseI to the petri dish. Gently swirl the dish and incubate at 37°C for 15-30 min.
2. Prepare and Digest Sample
Resuspend Cells
- Resuspend Cells and Pipette mix using a P1000 pipette set to 1000 µL in the petri dish.
- Transfer and filter the resuspensed mixture into a 15 mL conical through a 70 µm cell strainer.
- Wash the petri dish with 4 mL of cold 10mM EDTA. Transfer this liquid to the 15 mL conical and filter through the cell strainer as well.
- Centrifuge the cells at 1500 rpm at 4°C for 10 min
- Remove the supernatant, being careful not to disturb or aspirate the pellet.
- Resuspend the cell pellet in 200 µL of 0.04% BSA/PBS.
- Count on hemocytometer and automatic cell counter