TITLE: UPitt TriState SenNet TMC Human lung single cell suspension (test run)
AUTHORS: Marta Bueno, Lanping Guo, koenigshoffm, Oliver Eickelberg

[DESCRIPTION]
Human lung tissue dissociation protocol to produce single cell suspension that can be used for different purposes. Optimal incubation times to obtain cell suspension with great cell numbers and high cell viability from non-disease lungs.

[STEPS]
SECTION: Before you start
2. Same-day preparation:
  
 
 
  
  


SECTION: Before you start
2.1. Base media
    500 mL DMEM + 5 mL anti/anti (100x)

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2.2. Digestion media (10 mL per digestion -> scale accordingly)

I. Modified base media with Final DNAseI 100 µLper 10 mL (final 0.1mg/ml)

II. Then add corresponding proteases:
   
    II.a. Elastase / Collagenase (EC tubes)
    Final elastase                  250 µL per 10 mL (final 7.5U/ml) +
    Final collagenase IV       250 µL per 10 mL (final 400U/ml)

    II.b Liberase TL (TL tubes)
    Final Liberase TL            1 mLper 10 mL (final 0.25mg/ml)

SECTION: Tissue dissociation
3. Cut the lung tissue with a scalpel into approximately 3 cm3 pieces (size of a thumb) upon receipt. 
4. Place lung tissue into a 100mm petri dish. Place a ruler next to it and take pictures for archives.
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1.4.  :
    Stock of 5 mg in 2 mL of sterile ddH2O
    Aliquot in 500 µLin sterile conditions (1.25mg per aliquot)
    Store at -20 °C

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1.3.  :
    Stock of 100 mgin 1 mLof sterile ddH2O 
    The enzyme should be 0.22 microns filtered after reconstitution and prior to use 
    Aliquot in 500 µLin sterile conditions (150U per aliquot)
    Store at -20 °C

 :
    Stock of 1 g in 10 mLof sterile ddH2O
    Aliquot in 500 µLin sterile conditions (8000U per aliquot)
    Store at -20 °C

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1.2.    :
    Stock of 100 mg in 10 mL of sterile ddH2O 
    Aliquot in 300 µL in sterile conditions (3 mg per aliquot)
    Store at -20 °C

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1.1.  :
    Aliquot in 5 mL in sterile conditions.
    Store at -20 °C

SECTION: Before you start
1. Stock solutions 
Can be prepared beforehand & stored -20 °C:

3. Transfer them to the lid of a fresh 100mm petri dish. 
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4. Place lung tissue into a 100mm petri dish. Place a ruler/control object next to it and take pictures for archives.
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5. Transfer them to the lid of a fresh 100mm petri dish. 
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6. Each piece should be minced with scissors.
Locations: A-> Pleura; B-> bronchovascular bundle; C-> Parenchyma
(Label tubes accordingly)

 

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7. Transfer to a 50 ml conical Falcon tube with10 mL added digestion media. Use one tube per piece of tissue.

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8. Repeat the procedure with the remaining pieces.
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9. Incubate 30min at 37C, shaking 500rpm.
  

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10. Add 5 mL of FBS to the cell suspension to stop the digestion (final volume ~15ml)

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11. Filter through 100 μm cell strainer using the syringe plunger to press and smash the tissue, washing the tube and rinse the strainer with 5 mL of plain RPMI.

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12. Filter through 70 μm cell strainer using the syringe plunger to press and smash the tissue, washing tube and rinse the strainer with 5 mL of plain RPMI.

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13. Centrifuge. 300g, 15°C for 10 minutes.
300 x g, 10 min, 15 °C 

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14. Remove and discard the supernatant.
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15. Resuspend the cell pellets with 10 mL of plain RPMI and transfer to a fresh 15 ml Falcon conical tubes.

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15.1. Count cells here.  (This count can be skipped -not informative because of RBC numbers)
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15.2. To count: Take a 20 µL aliquot to determine total cell number, and mix with 20μl of VitaStain. Run AO/PI program in Nexcelom cell counter.
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16. Centrifuge. 300g, 15°C for 10 minutes.
300 x g, 10 min, 15 °C 

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17. Remove supernatant and resuspend the cell pellets with 5 mL of Red Blood Cell Lysis Buffer. Mix tubes by inversion and incubate for 1 min at Room temperature. 
(Note: It can be done by one after another)
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18. Fill up the tubes with 9 mL of plain RPMI.

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19. Centrifuge. 300g, 15°C for 10 minutes.
300 x g, 10 min, 15 °C 

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20. Remove and discard supernatant. Resuspend cell pellets with 10 mL of RPMI buffer kept on ice at all times.

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21. Centrifuge. 300g, 15°C for 10 minutes.
300 x g, 10 min, 15 °C 

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20.1. Count cells here: Take a 20 µL aliquot to determine total cell number, and mix with 20μl of VitaStain. Run AO/PI program in Nexcelom cell counter.
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22. Resuspend in the corresponding media for the next step
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22.1. For scRNA-seq: 
Resuspend in 500μl of PBS+2%FBS in the 15ml tube and then take 200μl of that suspension to a new Eppendorf tube. 
Pellet the cells by a centrifugation pulse, remove the supernatant and then resuspend to prepare ~4Million cells in 200μl of PBS+2%FBS (minimum 2M in 100μl)
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22.2. For storage: 
Resuspend in 500μl of PBS
Pellet the cells by a centrifugation pulse, remove the supernatant and stored at -80C