Seed IMR90 Cells (Day 1)
1. Pre-warm supplemented MEM growth medium (MEM + glutaMAX with 10% FBS, 1X penicillin/streptomycin, 1X NEAA, and 1X sodium pyruvate) and PBS in a 37°C water bath and a trypsin aliquot at room temperature (~30 minutes).
2. Trypsinize, collect, and pellet IMR90 cells at 1,000 x g at room temperature.
a. NOTE: Cells should be used on the third day after being plated at either 4.0 x 104 cells/mL.
3. Aspirate the supernatant and resuspend the cell pellet in 12 mL of supplemented growth medium. Triturate at least four times to thoroughly break up the majority of clumped cells. Add additional growth medium to dilute the cell suspension if desired (typically, a total volume of 12 mL per 2-3 15 cm plates collected).
4. Prepare a 1:1 dilution in Trypan Blue (50 μL Trypan blue + 50 μL cell suspension) in an Eppendorf tube.
5. Count cells that exclude Trypan Blue on a hemocytometer (count both sides of two independent loads, four counts total). Record the number of cells obtained per plate. Calculate the number of population doublings that occurred during the last passage (include the indicated values in the associated worksheet):
([(3.32(log(cells collected per plate)-log(cells plated per plate)) + previous APD])
6. Dilute cells to 1.0 x 105 cells/mL (for 6 well) or 1.25 x 105 cells/mL (for 12 well) in supplemented growth medium in a 50 mL tube or larger sterile bottle to the total volume that will be used for seeding (2 mL/well for 6 well and 800 μL/well for 12 well, making 2-5 mL extra).
7. Mix thoroughly by inversion and dispense the diluted cell suspension into each collagen-coated well in the appropriate volume:
a. 2 mL/well for 6 well plates
b. 800 μL/well for 12 well plates
8. Incubate in a humidified 37 °C incubator with 5% CO2 and 20% O2 for 1 day before combining with 16HBE cells.
Seed 16HBE Cells in Transwell Inserts (Day 1)
1. Pre-warm complete MEM growth medium (MEM + glutaMAX with 10% FBS and 1X penicillin/streptomycin) and PBS in a 37°C water bath and a trypsin aliquot at room temperature (~30 minutes).
2. Trypsinize, collect and pellet 16HBE cells at 1,000 x g at room temperature.
a. NOTE: Cells should be used on the third day after being plated at 1.75 x 105 cells/mL.
3. Aspirate the supernatant and resuspend the cell pellet in 12 mL of complete growth medium. Triturate at least four times to thoroughly break up the majority of clumped cells. Add additional growth medium to dilute the cell suspension if desired (typically, a total volume of 12 mL per 1-2 15 cm plates collected).
4. Prepare a 1:5 dilution in PBS (800 μL PBS + 200 μL cell suspension) in an Eppendorf tube.
5. Count cells on a hemocytometer (count both sides of two independent loads, four counts total). Record the number of cells obtained per plate. Calculate the number of population doublings that occurred during the last passage (include the indicated values in the associated worksheet):
([3.32(log(cells collected per plate)-log(cells plated per plate)) + previous APD])
6. Dilute cells to 8.0 x 105 cells/mL (for both 24mm and 12mm inserts) in complete growth medium in a 50 mL tube or larger sterile bottle to the total volume that will be used for seeding (2 mL/24mm insert and 500 μL/12mm insert, making 2-5 mL extra).
7. Mix thoroughly by inversion and dispense the diluted cell suspension into each collagen-coated insert in the appropriate volume:
a. 2 mL/insert for 24mm inserts
b. 500 μL/insert for 12mm inserts
8. Dispense complete growth medium to the basolateral compartment of each well in the appropriate volume:
a. 2 mL/well for 24mm inserts (6 well)
b. 800 μL/well for 12mm inserts (12 well)
c. NOTE: Check that there are no air bubbles trapped underneath insert membrane.
9. Incubate in a humidified 37°C incubator with 5% CO2 and 20% O2 for 1 day until combining with IMR90 cells.
Combine Cells (Day 2)
Cells should be combined approximately 24 hours before exposure.
1. Aspirate the medium from the basolateral compartment of the wells containing Transwell inserts seeded with 16HBE cells.
a. NOTE: Do not aspirate the medium from the apical compartment.
2. Carefully transfer Transwell inserts with 16HBE cells into the wells containing IMR90 cells without changing the culture medium on top of either cell type.
a. NOTE: Check that there are no air bubbles trapped underneath insert membrane.
3. Incubate in a humidified 37°C incubator with 5% CO2 and 20% O2 for 1 day until exposure to diesel exhaust particulates (DEP).
Exposure and Harvest (Day 3)
1. Pre-warm basal medium (MEM + glutaMAX with 1X penicillin/streptomycin) in a 37°C water bath.
2. Thaw frozen PM preparation and dilute to desired concentration in basal medium. Vortex thoroughly.
3. Carefully aspirate the medium from the apical compartment of the Transwell inserts and replace with either vehicle (basal medium) or diluted PM suspension in the appropriate volume:
i. 1 mL/insert for 24mm inserts (6 well)
ii. 250 μL/insert for 12mm inserts (12 well)
iii. NOTE: Vortex PM suspension occasionally to mix the settling particles.
4. Return to the humidified 37°C incubator for the desired exposure duration.
5. Harvest VEH and PM exposure samples for RNA or whole cell protein immediately after desired exposure duration.
a. 16HBE RNA Harvest
i. Remove Transwell inserts from the exposure plate, blot basal surfaces with Kimwipes, and transfer into new, clean wells.
ii. Carefully aspirate the medium from the apical compartment.
iii. Rinse the apical surface with 1 mL (24mm inserts) or 500 μL (12mm inserts) of PBS. Aspirate the PBS wash.
iv. Add 300 μL of RNA lysis buffer (with 1% 2-mercaptoethanol), pipette up and down while distributing the lysis buffer across the membrane surface, and transfer the lysate from each sample to individual Eppendorf tubes.
• NOTE: Use cell scrapers to dislodge cells for 24mm inserts.
v. Store at -80°C until RNA isolation.
b. IMR90 RNA Harvest
i. Carefully aspirate the medium from the basolateral compartment.
ii. Add 300 μL (for 6 well) or 200 μL (for 12 well) of RNA lysis buffer (with 1% 2-mercaptoethanol), pipette up and down while distributing the lysis buffer across the well surface, and transfer the lysate from each sample to individual Eppendorf tubes.
• NOTE: Use cell scrapers to dislodge cells for 6 well plates.
iii. Store at -80°C until RNA isolation.
c. Protein Harvest