TITLE: Fluorescent Gelatin Degradation Assay to Evaluate EVh Action in TME Cells
AUTHORS: Bianca Cruz Pachane, Heloisa Sobreiro Selistre de Araujo

[DESCRIPTION]
The fluorescent gelatin degradation assay is a method to study cell invasion by detecting gelatinase activity in vitro upon epifluorescence microscopy analysis. In this protocol, the method has been applied to evaluate the effect of hypoxic EVs from TNBC cell line MDA-MB-231 in four cellular models for the tumor microenvironment - MDA-MB-231 (tumor cell), HUVEC (endothelial cell), HDFa (dermal fibroblast) and THP-1 (monocyte). Adapted from Pachane et al (2022) (PMID: 36293503).

[BEFORE_START]
Fluorescent gelatin preparation: Under sterile conditions, solubilize the fluorescent gelatin stock at 37 °C with warmed PBS following the manufacturer's instructions for a concentration of 5 mg/mL. Aliquot in microtubes and maintain at -20 ºC until time of use. 
Before use, thaw gelatin at 37 °C for 30 min . Dilute stock to a 0.2 mg/mL working solution with warmed PBS and maintain at 37 °C until use. 

Cell culture: Maintain cells in culture during at least two passages after thawing.


[STEPS]
SECTION: Fluorescent Gelatin Coating
1. Open a new 96-well black plate under sterile conditions and label groups in technical triplicates to contain a vehicle (PBS) control (i.e., untreated cells in OptiMEM) and the EVh-treated group (i.e., EVh-treated cells in OptiMEM) for each cell line.
SECTION: Fluorescent Gelatin Coating
1.1. Experimental plate map:
 

SECTION: Fluorescent Gelatin Coating
2. Apply 70 µL of the fluorescent gelatin working solution 0.2 mg/mL directly to the bottom of each well and prevent the formation of bubbles. 
SECTION: Fluorescent Gelatin Coating
3. Incubate plate for 30 min at 37 °C .

SECTION: Fluorescent Gelatin Coating
4. Carefully remove excess coating (avoid touching well bottom).
SECTION: Fluorescent Gelatin Coating
5. Pre-condition coating with 200 µL for 30 min at 37 °C .

SECTION: Cell seeding
6. Subculture cells as usual. Resuspend cell pellet in OptiMEM and count cells using the trypan blue exclusion method.
SECTION: Cell seeding
7. Remove pre-conditioning media from the wells (avoid touching well bottom).
SECTION: Cell seeding
8. Add cell suspension into each well to a total volume of 200 µl:
MDA-MB-231:  5x103 cells/well (= 1x105 células/ml)
HUVEC: 5x103 cells/well (= 1x105 células/ml)
HDFa: 2x103 cells/well (= 1x104 células/ml)
THP-1: 5x103 cells/well (= 1x105 células/ml)
SECTION: Cell seeding
10. Incubate plate for 1440 min at 37 °C 

SECTION: Fixation and Cell Staining
11. Remove the supernatant by aspiration.
SECTION: Fixation and Cell Staining
12. Fix cells with 100 µL warmed 4% PFA at Room temperature for 10 min

SECTION: Fixation and Cell Staining
13. Wash wells twice with 100 µL 

SECTION: Fixation and Cell Staining
14. Permeabilize cells with 100 µL  at Room temperaturefor 5 min 

SECTION: Fixation and Cell Staining
15. Wash wells twice with 100 µL

SECTION: Fixation and Cell Staining
16. Stain cells with the DAPI + Phalloidin-647 mixture. Add 100 µL to each well and incubate at Room temperature for 20 min .
SECTION: Fixation and Cell Staining
17. Wash wells twice with 100 µL 
SECTION: Fixation and Cell Staining
18. Maintain wells with 200 µL for analysis. Seal the plate with   and cover it with aluminum foil for storage at 4 °C for up to 6 months.

SECTION: Cell Imaging by Epifluorescence HTS
19. Using the microscope ImageXpress Micro XLS+ (Molecular Devices), check the template for the Corning 3603 plate and the filters for DAPI (nuclei), FITC (gelatin) and Cy5 (phalloidin-647).
SECTION: Cell Imaging by Epifluorescence HTS
20. Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
SECTION: Cell Imaging by Epifluorescence HTS
21. Check the wells using the 4X objective.
SECTION: Cell Imaging by Epifluorescence HTS
22. Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
SECTION: Cell Imaging by Epifluorescence HTS
23. Acquire the plate. Export metadata for analysis.
SECTION: Cell Imaging by Epifluorescence HTS
24. For representative images, change into the 40x objective and adjust the laser focus. 
Select the sites of interest and acquire. 
Export image channels and combinations.
SECTION: Gelatin Degradation Quantification on FIJI
25. On FIJI (ImageJ), import HTD files through BioFormats.
SECTION: Gelatin Degradation Quantification on FIJI
26. Images should already be scaled. If not, adjust scale based on the objective lens used for acquisition.
SECTION: Gelatin Degradation Quantification on FIJI
27. Set measurements to contain "Area", "Standard Deviation", "Shape Descriptor", "Mean grey value", "Perimeter" and "Display label".
SECTION: Gelatin Degradation Quantification on FIJI
28. Concatenate all stacks into a single hyperstack.
SECTION: Gelatin Degradation Quantification on FIJI
29. Split channels and select the FITC stack for analysis.
SECTION: Gelatin Degradation Quantification on FIJI
30. Set a threshold to encompass the degradation spots but not the background. Write down the threshold values. Create a new stack with the binary images. 
SECTION: Gelatin Degradation Quantification on FIJI
31. To measure the degraded area, analyze particles with a range of "5-Infinity" and select "Summarize".
SECTION: Gelatin Degradation Quantification on FIJI
32. Save CSV file. The degraded area (in µm2) per site will be compared between groups in the statistical analysis.
SECTION: Cell Counting on FIJI
33.   
SECTION: Cell Counting on FIJI
34. Split channels and select the DAPI stack for counting.
SECTION: Cell Counting on FIJI
35. Set a threshold to contain nuclei. Create a new stack with the binary images.
SECTION: Cell Counting on FIJI
36. To count cells, analyze particles with a range of "10-infinity" and select "Summarize". 
SECTION: Cell Counting on FIJI
37. Save CSV file. 
SECTION: Cell Morphology Analysis on FIJI
39. Split channels and select the Cy5 stack for analysis.
SECTION: Cell Morphology Analysis on FIJI
38.   
SECTION: Cell Morphology Analysis on FIJI
40. Duplicate the stack as a guide.
SECTION: Cell Morphology Analysis on FIJI
41. Set a threshold to encompass cell cytoplasm. Create a new stack with the binary images.
SECTION: Cell Morphology Analysis on FIJI
42. Using the duplicated stack as a guide, section cells using the "pencil" tool with a 3 px thickness.
SECTION: Cell Morphology Analysis on FIJI
43. To analyze cell morphology, analyze particles with a range of "10-Infinity" and check "Clear Results".
SECTION: Cell Morphology Analysis on FIJI
44. Save CSV file. The cell circularity index of each cell will be compared between groups in the statistical analysis.
SECTION: Image Processing for Representative Cells
48. Select the cell of interest in a 200x200 px squared selection.
SECTION: Image Processing for Representative Cells
45.   
SECTION: Image Processing for Representative Cells
46. Split the channels of the stacks of interest.
SECTION: Image Processing for Representative Cells
47. Adjust channel colors using the "Lookup Tables" menu.
SECTION: Image Processing for Representative Cells
49. Save selections in each channel and the combination of all channels in PNG images.
SECTION: Cell seeding
9. Treat cells with EVh (109 particles/ml) or the equivalent treatment volume in PBS.