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Protocols
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Cell Digest for Tissue Samples stored in CryoStore (CS) 10

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

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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
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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.

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R10 Solution
  1. Set up 37°C Water Bath to thaw any reagents frozen at -20°C
  2. Gather the following:
    1. RPMI 1640
    2. 50 mL FBS aliquot
    3. 5 mL Pen/Strep aliquot
    4. 5 mL L-Glutamine aliquot
  3. Thaw frozen reagents for 10 min or until completely thawed.
  4. 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).
  5. Combine all reagents in the top of a Vacuum Filter bottle.
  6. Slowly turn on the vacuum line to begin filtering.
  7. Once all liquid has been filtered, carefully remove the top of the bottle and discard the top portion and screw on the included cap.
  8. Invert to mix R10 solution.
  9. Store at 4°C
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R10 + Collagenase P Solution
  1. Gather the following:
    1. R10 Solution
    2. 100 mg Collagenase P
  2. The Collagenase P will ship as 100 mg, lyophilized in a small class container with a rubber stopper. Do not remove the rubber stopper!
  3. Using an aliquot of R10 Solution, aspirate 1 mL of R10 using a sterile needle and syringe.
  4. Puncture the syringe through the rubber stopper and dispense 1 mL of R10 solution into the glass container
  5. Invert to mix until the collagensase is completely dissolved.
  6. Using the same needle, aliquot 100 uL (0.1 mL) if conducting the cell digest the same day. Otherwise store at 4°C.
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10mM EDTA
  1. Gather the following:
    1. 0.5M EDTA
    2. 1x PBS
  2. Combine 1 mL of 0.5M EDTA in 49 mL of 1x PBS in a new 50 mL Conical
  3. Store at 4°C.
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10mM EDTA
  1. Gather the following:
    1. BSA
    2. 1x PBS
  2. Combine 2µL of BSA in 25 mL of 1x PBS in a new 50 mL Conical
  3. Store at 4°C.

2. Prepare Sample for Digest

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Thaw and Prepare Tissue
  1. If water bath is not set up, prepare a 37°C water bath
  2. Acquire tissue sample(s) from -80°C Freezer.
  3. Incubate sample for 10 min at 37°C.
  4. Move sample to biosafety hood that contains the following:
    1. 4 mL of 1x PBS
    2. Sterile Petri dish
    3. Scalpel/Scissors
    4. R10 solution
    5. R10 + Collagenase P aliquot
  5. Wash sample in tube with 1x PBS for a total of 3 washes.
    1. Remove CS 10 solution
    2. Add 1 mL of 1xPBS to tube containing tissue sample
    3. Remove 1 mL of supernatant
    4. Repeat steps b-c for a total of 3 washes
  6. Transfer tissue sample to the Petri dish. Use a clean P1000 pipette tip if necessary to help remove the sample from the tube
  7. Cut the skin into small pieces using the scalpel and scissors. Tissue will be very tough!
  8. 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.
    1. 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
  9. Incubate the petri dish in an incubate at 37°C. (The Scott Lab has one directly next to the biosafety hood in their tissue culture room).