TITLE: Preparation of a Single Cell Suspension from Bronchoalevolar Lavage
AUTHORS: Steven B. Wells, Peter A. Szabo, Basak Ural

[DESCRIPTION]
This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

[STEPS]
SECTION: Preparing Mediums and Buffers
1. Create the following IMDM-FBS-PSQ Media in a 500 mL bottle of IMDM by using the table below: 
            Component  Volume (mL)  Starting Conc.  Final Conc.*    IMDM  500  -  -    Penicillin-Streptomycin-Glutamine  5  100X  1X    FBS  50  100%  10%  
 

SECTION: Preparing Mediums and Buffers
2. Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS without calcium and magnesium by using the table below:
 
            Component  Volume (mL)  Starting Conc.  Final Conc.*    DPBS  500  -  -    FBS  25  100%  5%    EDTA  50  0.5M  1mM  
 
SECTION: Performing the Lavage
3. Identify, and using a scissors, make an incision in one of the secondary bronchi that connects to the lower lobes of the left lung.
SECTION: Performing the Lavage
4. Insert the catheter about 5 to 10 centimeters into the incision, remove the needle and attach a 3- way Stopper to the catheter.
SECTION: Performing the Lavage
5. Fill a 50 mL syringe with PBS and connect to the 3-way Stopper.

SECTION: Performing the Lavage
6. Slowly inject 25 mL of cold PBS into the lungs. Watch the lungs inflate, and do not overinflate.

SECTION: Performing the Lavage
7. Attach an empty 25 mL syringe to the final spot of the 3-way Stopper.

SECTION: Performing the Lavage
8. Collect about 10 mL of BAL fluid (BALF) from lungs.

SECTION: Performing the Lavage
9. Repeat the previous steps until 50 mL of PBS is injected into the lungs and at least 25 mL to 50 mL of BALF is collected.

SECTION: Processing the BALF
10. Spin for 5 min at 400 x g at 4 °C, remove and save the supernatant in 2 mL cyrovials – record supernatant volume saved below:
_________ mL
SECTION: Processing the BALF
11. Resuspend the cell pellet in 10 mL of IMDM, add 10 µL of benzonase to the BALF and at 37 °C for 30 min.

SECTION: Processing the BALF
12. Add 40 mL of IMDM (NO ADDITIVES) to the cell suspension, spike in 0.500 mL of EDTA 0.5 Molarity (M) pH 8.0.

SECTION: Ficoll-Paque
13. Filter the cell suspension through a 100 micromolar (µM) cell strainer.

SECTION: Ficoll-Paque
14. In two 50 mL tubes, layer 25 mL of cell suspension on top of 15 mL of Ficoll-Paque Media PLUS.

SECTION: Ficoll-Paque
15. Spin for 20 min, 1200 x g at 20 °C with 4 acceleration and 0 brake, evenly distribute the tubes across the entire rotor to prevent wobbling (use all four buckets if possible as opposed to just two).

SECTION: Ficoll-Paque
16. Remove the mononuclear cell layer from both tubes with a transfer pipet and combine in one 50 mL tube. Add cold DPBS-FBS-EDTA Solution to a final volume of 50 mL and centrifuge the cell suspension for 10 min at 400 x g, 4 °C.

SECTION: Ficoll-Paque
17. Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 10 min at 120 x g, 4 °C.

SECTION: Ficoll-Paque
18. Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.

SECTION: Cell Count
19. Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: __________ cells/mL, __________ %viable
final volume: __________ mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: __________
SECTION: Freeze-down and QC
20. (Optional QC) Aliquot 2 x 106 cells to a 5mL Falcon tube and place on ice for subsequent flow cytometric analysis.
SECTION: Freeze-down and QC
21. Aliquot cells for analysis or experimentation, and then freeze down cells in up to 2 x 107 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a -80 °C freezer (1 mL-1.5 mL aliquots, round down to the nearest 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.