Materials and Reagents

6-well cell culture plates (Corning, Costar^®, catalog number: 3516 )
Normal human fibroblasts (IMR-90) (ATCC, catalog number: CCL-186 )
DMEM
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 26140095 )
Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Amphotericin B (Thermo Fisher Scientific, Gibco^TM, catalog number: 15290018 )
RPMI 1640 medium, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 11835030 )
DPBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190144 )
NP-40
Sodium chloride (NaCl)
Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
Phosphatase inhibitor (EMD Millipore, catalog number: 524625 )
α-SMA antibody (American Research Products, catalog number: 03-61001 )
β-actin antibody (Sigma-Aldrich, catalog number: A5441 )
Tween 20
Fibroblast culture media (see Recipes)
Lysis buffer (see Recipes)

Equipment

Cell culture incubator, 37 °C, 5% CO₂: 95% air atmosphere (Thermo Fisher Scientific, Forma^TM, model: Direct Heat CO₂ Incubator )

Procedure

Seed fibroblasts at a density of 1 x 10⁶ per well of a 6-well cell culture plate in a total volume of 1 ml. Allow cells to adhere to cell culture plate (2-6 h).
Harvest bronchoalveolar lavage (BAL) fluid from mice (Han and Ziegler, 2013). Mice can be treated with bleomycin (1.75 U/kg) or saline as a negative control. Determine the protein concentration of bronchoalveolar lavage (BAL) fluid. Using RPMI 1640 media, normalize all BAL samples to the same protein concentration in a total volume of 1 ml.
Notes:

Typically, 0.5-1 ml of BAL fluid is used and RPMI is used to normalize the protein concentration to a final total volume of 1 ml.
Alternatively, this assay can be performed in vitro using conditioned media from macrophage cell lines or bone marrow derived macrophages. Harvest the conditioned media from macrophages post-transfection or post-treatment, using 1 million cells per 1 ml of media.

Remove media from fibroblasts and rinse with 1.5 ml room temperature 1x PBS, add normalized BAL fluid to fibroblasts.

Incubate fibroblasts with BAL fluid for 24 h in a cell culture incubator, 37 °C, 5% CO₂: 95% air atmosphere.
Harvest fibroblasts by lysing cells in lysis buffer (see Recipes).

Determine α-SMA expression by immunoblot analysis. The expression of α-SMA protein was determined by Western blotting using 20 μg of total cellular protein. After blocking in 5% milk, the membranes were probed with mouse α-SMA primary antibody using a 1:3,000 dilution in Tris buffered saline containing 0.1% Tween 20, the membranes were incubated with the anti-mouse secondary antibody (1:2,000) in antibody dilution buffer for 1 h.

Data analysis

Results can be shown as a representative immunoblot (Figure 1); also see Larson-Casey et al. (2016).

Figure 1. Immunoblot analysis of α-SMA in IMR-90 fibroblasts cultured in BAL fluid (BALF) from saline or bleomycin (Bleo) exposed WT mice

Recipes

Fibroblast culture media
DMEM
10% heat-inactivated FBS
100 U/ml, 100 μg/ml penicillin-streptomycin
1.25 μg/ml amphotericin B (fungizone)
Lysis buffer
1% NP-40
0.15 M NaCl
0.05 M Tris pH 7.4
1 protease tablet
Phosphatase inhibitor diluted 1:100

Acknowledgments

This work was supported by 2R01ES015981 & VA merit review BX001135.

References

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