Primary Lung Fibroblasts; Normal, Human Item ID: ATCPCS201013

1. Obtain one vial of Primary Lung fibroblast cells ; Normal, Human (ATCC PCS-201-013) from the liquid nitrogen tank; make sure that the caps of all components are tight.
2. Thaw the components of Fibroblast Growth Kit-Low serum ATCC® PCS-201-041™just prior to adding them to the Fibroblast Basal Medium ATCC® PCS-201-030™ .
3. Obtain one bottle of Fibroblast Basal Medium ATCC® PCS-201-030™ from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique and working in a laminar flow hood or biosafety cabinet, transfer the indicated volume of each growth kit component, as indicated in Table 1, into the bottle of basal medium using a separate sterile pipette for each transfer.

Table 1. Fibroblast Growth Kit-Low Serum Components

Component	Volume	Final Concentration
rh FGF b	0.5 mL	5 ng/mL
L-glutamine	18.75 mL	7.5 mM
Ascorbic acid	0.5 mL	50 µg/mL
Hydrocortisone Hemisuccinate	0.5 mL	1 µg/mL
rh Insulin	0.5 mL	5 µg/mL
Fetal Bovine Serum	10.0 mL	2%

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.

1. Passage normal lung fibroblast cells when culture has reached approximately 80% to 90% confluence, and are actively proliferating.
2. Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Briefly rinse the cell layer with 3 to 5 mL DPBS (ATCC 30-2200) to remove residual traces of serum and then aspirate and discard the DPBS.
5. Add pre-warmed trypsin-EDTA solution (2 to 3 mL for every 25 cm2) to each flask.
6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells.
7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
8. When the majority of cells are detached, quickly add an equal volume of Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
10. Add 3 to 5 mL Trypsin Neutralizing Solution (ATCC PCS-999-004) to the flask to collect any remaining dissociated cells. Transfer remaining cells into the centrifuge tube.
11. Repeat steps 10 as needed until all cells have been collected from the flask.
12. Centrifuge the cells at 150 x g for 3 to 5 minutes.
13. Carefully aspirate the neutralized dissociation solution from the cell pellet and re-suspend the cells in 5 to 8 mL fresh, pre-warmed, complete growth medium.
14. Count the cells and seed new flasks at a density of 2,500 to 5,000 cells per cm2.
15. Place freshly seeded flasks in a 37°C, 5% CO2, incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.