Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression

Materials and Reagents

1. 6-well cell culture plates (Corning, Costar^®, catalog number: 3516 )
   
2. Normal human fibroblasts (IMR-90) (ATCC, catalog number: CCL-186 )
   
3. DMEM
   
4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 26140095 )
   
5. Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
   
6. Amphotericin B (Thermo Fisher Scientific, Gibco^TM, catalog number: 15290018 )
   
7. RPMI 1640 medium, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 11835030 )
   
8. DPBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190144 )
   
9. NP-40
   
10. Sodium chloride (NaCl)
    
11. Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
    
12. Phosphatase inhibitor (EMD Millipore, catalog number: 524625 )
    
13. α-SMA antibody (American Research Products, catalog number: 03-61001 )
    
14. β-actin antibody (Sigma-Aldrich, catalog number: A5441 )
    
15. Tween 20
    
16. Fibroblast culture media (see Recipes)
    
17. Lysis buffer (see Recipes)
    

Equipment

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

Procedure

1. 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).
   
2. 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:
1. 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.
   
2. 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.
   
3. Remove media from fibroblasts and rinse with 1.5 ml room temperature 1x PBS, add normalized BAL fluid to fibroblasts.
   
1. Incubate fibroblasts with BAL fluid for 24 h in a cell culture incubator, 37 °C, 5% CO₂: 95% air atmosphere.
   
2. Harvest fibroblasts by lysing cells in lysis buffer (see Recipes).
   
4. 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

1. Fibroblast culture media
   DMEM
   10% heat-inactivated FBS
   100 U/ml, 100 μg/ml penicillin-streptomycin
   1.25 μg/ml amphotericin B (fungizone)
   
2. 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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