# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 2 Materials and Methods
### Analysis of Cell Adhesion to Extracellular Matrix Components  
Two ELISA-based approaches were used to assess _S. schenckii_ adhesion to extracellular matrix (ECM) components, as previously reported [59]. Nunc MaxiSorp(tm) flat-bottom 96-well microplates were coated with 1 \(\upmu\)g of human laminin, human elastin, human fibrinogen, recombinant human fibronectin, recombinant human thrombospondin-1, human type-I collagen, or bovine type-II collagen (all from Sigma-Aldrich). The proteins were suspended in 0.05% (\(w/v\)) PBS Tween 20, and plates were incubated for 3 h at room temperature. Then, plates were blocked overnight with 1% (\(w/v\)) bovine serum albumin (BSA) in PBS at 4 \({}^{\circ}\)C. Finally, plates were washed three times with 0.05% (\(w/v\)) PBS Tween 20 and were immediately used in adhesion experiments.  
Aliquots of 100 \(\upmu\)L containing 5 \(\times\) 10\({}^{6}\) yeast-like cells were added to each well, and plates were incubated for 60 min at 37 \({}^{\circ}\)C. Plates were washed three times with 0.05% (\(w/v\)) PBS Tween 20, 100 \(\upmu\)L of polyclonal anti-rGp70 [56] at a working dilution 1:3000 was added, and plates were incubated 2 h at room temperature. Then, plates were washed again, and 100 \(\upmu\)L of goat anti-rabbit IgG-peroxidase antibody (1:5000; Sigma-Aldrich) was added and further incubated for 2 h at room temperature. Finally, the presence of peroxidase was revealed by incubating at room temperature for 20 min with 0.1 mg mL\({}^{-1}\) 2,2\({}^{\prime}\)-Azino-bis(3-ethylbenzothazoline-6-sulfonic acid) diammonium salt and 0.006% (\(v/v\)) hydrogen peroxide and stopped by adding 2 N sulfuric acid. The absorbance at 450 nm was read in a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). When required, fungal cells were incubated with either polyclonal antibodies against rHsp60 or rPap1 at a working dilution of 1:3000 for 60 min at 37 \({}^{\circ}\)C. Then, cells were washed three times with PBS and used in the adhesion assays.  
Alternatively, bacteria expressing the recombinant proteins were washed three times with PBS, resuspended in 5 mL of the same buffer added with 50 mg mL\({}^{-1}\) lysozyme (Sigma-Aldrich), and incubated for 60 min at 37 \({}^{\circ}\)C. Then, 1% (\(w/v\)) SDS was added, and cells were disrupted by incubating for 30 min at 37 \({}^{\circ}\)C and shaking for 20 min in a vortex. The cell homogenate was centrifuged at 21,000\(\times\)\(g\) at 4 \({}^{\circ}\)C, then 5 \(\upmu\)g protein from the supernatant were added to wells coated with the ECM proteins, incubated 60 min at 37 \({}^{\circ}\)C, and washed three times with 0.05% (\(w/v\)) PBS Tween 20. Polyclonal antibodies against rHsp60 or rPap1 at a working dilution of 1:3000 were added and incubated 2 h at room temperature. The secondary goat anti-rabbit IgG-peroxidase antibody and detection of peroxidase activity were performed as above described.  
In both formats, wells coated only with the blocking agent (BSA) were used as control, and the readings from these wells were subtracted from the values obtained from wells coated with ECM proteins.