Preparation of a Single Cell Suspension from Bronchoalevolar Lavage

Create the following IMDM-FBS-PSQ Media in a 500mL bottle of IMDM by using the table below:

A	B	C	D
Component	Volume (mL)	Starting Conc.	Final Conc.*
IMDM	500	-	-
Penicillin-Streptomycin-Glutamine	5	100X	1X
FBS	50	100%	10%

Table 1.*Final Concentration is approximate.

Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS without calcium and magnesium by using the table below:

A	B	C	D
Component	Volume (mL)	Starting Conc.	Final Conc.*
DPBS	500	-	-
FBS	25	100%	5%
EDTA	50	0.5M	1mM

Table 2.*Final Concentration is approximate.

Identify, and using a scissors, make an incision in one of the secondary bronchi that connects to the lower lobes of the left lung.

Insert the catheter about 5 to 10 centimeters into the incision, remove the needle and attach a 3- way Stopper to the catheter.

Fill a 50mL syringe with PBS and connect to the 3-way Stopper.

Slowly inject 25mL of cold PBS into the lungs. Watch the lungs inflate, and do not overinflate.

Attach an empty 25mL syringe to the final spot of the 3-way Stopper.

Collect about 10mL of BAL fluid (BALF) from lungs.

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

Spin for 0h 5m 0s at 400x g,0h 0m 0s at 4°C, remove and save the supernatant in 2mL cyrovials – record supernatant volume saved below:

_________ mL

Resuspend the cell pellet in 10mL of IMDM, add 10µL of benzonase to the BALF and at 37°C for 0h 30m 0s.

Add 40mL of IMDM (NO ADDITIVES) to the cell suspension, spike in 0.500mL of EDTA 0.5Molarity (M) 8.0.

Filter the cell suspension through a 100micromolar (µM) cell strainer.

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

Spin for 0h 20m 0s, 1200x g,0h 0m 0s 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).

Remove the mononuclear cell layer from both tubes with a transfer pipet and combine in one 50mL tube. Add cold DPBS-FBS-EDTA Solution to a final volume of 50mL and centrifuge the cell suspension for 0h 10m 0s at 400x g,0h 0m 0s, 4°C.

Remove the supernatant and re-suspend the cell pellet in 50mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 0h 10m 0s at 120x g,0h 0m 0s, 4°C.

Remove the supernatant and re-suspend the cell pellet in cold 10mL IMDM-FBS-PSQ Media.

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: __________

(Optional QC) Aliquot 2 x 10⁶ cells to a 5mL Falcon tube and place on ice for subsequent flow cytometric analysis.

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 (1mL-1.5mL aliquots, round down to the nearest 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.