UPitt TriState SenNet TMC Human lung single cell suspension (test run)

Stock solutions

Can be prepared beforehand & stored -20°C:

:

Aliquot in `5mL` in sterile conditions.



Store at `-20°C`

mg :

Stock of `100mg` in `10mL` of sterile ddH<sub>2</sub>O 



Aliquot in `300µL` in sterile conditions (`3mg` per aliquot)



Store at `-20°C`

:

Stock of `100mg`in `1mL`of sterile ddH<sub>2</sub>O 



The enzyme should be 0.22 microns filtered after reconstitution and prior to use 

Aliquot in `500µL`in sterile conditions (150U per aliquot)



Store at `-20°C`

:

Stock of `1g` in `10mL`of sterile ddH<sub>2</sub>O



Aliquot in `500µL`in sterile conditions (8000U per aliquot)



Store at `-20°C`

:

Stock of `5mg` in `2mL` of sterile ddH<sub>2</sub>O



Aliquot in `500µL`in sterile conditions (1.25mg per aliquot)



Store at `-20°C`

Same-day preparation:

Base media

`500mL` DMEM + `5mL` anti/anti (100x)

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

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

II. Then add corresponding proteases:

II.a. Elastase / Collagenase (EC tubes)

Final elastase                  `250µL` per `10mL` (final 7.5U/ml) +



Final collagenase IV       `250µL` per `10mL` (final 400U/ml)





II.b Liberase TL (TL tubes)

Final Liberase TL            `1mL`per `10mL` (final 0.25mg/ml)

Cut the lung tissue with a scalpel into approximately 3 cm³ pieces (size of a thumb) upon receipt.

Place lung tissue into a 100mm petri dish. Place a ruler/control object next to it and take pictures for archives.

Transfer them to the lid of a fresh 100mm petri dish.

Each piece should be minced with scissors.

Locations: A-> Pleura; B-> bronchovascular bundle; C-> Parenchyma

(Label tubes accordingly)

A	B
EC-A	TL-A
EC-B	TL-B
EC-C	TL-C

Table 1. Samples generated.

Transfer to a 50 ml conical Falcon tube with10mL added digestion media. Use one tube per piece of tissue.

Repeat the procedure with the remaining pieces.

Incubate 30min at 37C , shaking 500rpm.

500rpm

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

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

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

Centrifuge. 300g, 15°C for 10 minutes.

300x g,15°C

Remove and discard the supernatant.

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

Count cells here. (This count can be skipped -not informative because of RBC numbers)

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.

Centrifuge. 300g, 15°C for 10 minutes.

300x g,15°C

Remove supernatant and resuspend the cell pellets with 5mL of Red Blood Cell Lysis Buffer. Mix tubes by inversion and incubate for 0h 1m 0s at Room temperature.

(Note: It can be done by one after another)

Fill up the tubes with 9mL of plain RPMI.

Centrifuge. 300g, 15°C for 10 minutes.

300x g,15°C

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

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.

Centrifuge. 300g, 15°C for 10 minutes.

300x g,15°C

Resuspend in the corresponding media for the next step

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)

For storage:

• Resuspend in 500μl of PBS
• Pellet the cells by a centrifugation pulse, remove the supernatant and stored at -80C