TITLE: Cell Invasion in Direct Co-Culture
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 a direct co-culture assay, using four cellular models for the tumor microenvironment. Hence, MDA-MB-231 (tumor cell), HUVEC (endothelial cell), HDFa (dermal fibroblast), and THP-1 (monocyte) have been seeded in six distinct combinations. 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 °C until time of use. 
Before use, thaw gelatin at 37 °Cfor 30 min. Dilute stock to a 0.2 mg/mLworking solution with warmed PBS and maintain at 37 °Cuntil use. 

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

[STEPS]
SECTION: Experimental Design and Plate 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 co-culture combination:
MDA-MB-231 + HUVEC
MDA-MB-231 + HDFa
MDA-MB-231 + THP-1
HUVEC + HDFa
HUVEC + THP-1
HDFa + THP-1
2. 
 

3. Apply 70 µL of the fluorescent gelatin working solution at 0.2 mg/mL directly to the bottom of each well. 
Avoid the formation of bubbles.

4. Incubate plate for 30 min at 37 °C 

5. Carefully remove excess coating by aspiration. 
Avoid touching well bottom.
6. Pre-condition coating with 200 µL  for 30 min at 37 °C 

SECTION: Cell Staining and Seeding
7. Subculture cells as usual. Resuspend cell pellets in OptiMEM and count cells using the trypan blue exclusion method.
SECTION: Cell Staining and Seeding
8. Stain cells using CellTrace™ CSFE or CellTracker™ CMPTX
SECTION: Cell Staining and Seeding
8.1. Separate each cell line in two 15-ml conical tubes, each containing 1x106 cells in 1 mL 

SECTION: Cell Staining and Seeding
8.2. Add 1 µL or 0.5 µL to cell suspensions, to achieve a 5 µM final concentration. Pipette well to mix.
SECTION: Cell Staining and Seeding
8.3. Incubate cell suspensions at 37 °C for 30 min

SECTION: Cell Staining and Seeding
8.4. Dilute cell suspensions with 4 mL and centrifuge at 1200 rpm, 5 min 

SECTION: Cell Staining and Seeding
8.5. Resuspend cell pellets in 1 mL and recount cells using the trypan blue exclusion method.

SECTION: Cell Staining and Seeding
9. Remove the pre-conditioning media from the plate.
SECTION: Cell Staining and Seeding
10. Add cell suspensions to wells following the table below:

 

SECTION: Cell Staining and Seeding
11. Treat cells with EVh (109 particles/ml) or the equivalent treatment volume in PBS.
SECTION: Cell Staining and Seeding
12. Incubate plate for1440 minat 37 °C 

SECTION: Fixation and Cell Staining
13. Remove the supernatant by aspiration.
SECTION: Fixation and Cell Staining
14. Fix cells with 100 µLwarmed 4% PFA at Room temperaturefor 10 min
SECTION: Fixation and Cell Staining
15. Wash cells twice with 100 µL 

SECTION: Fixation and Cell Staining
16. Permeabilize cells with 100 µLat Room temperature for 5 min

SECTION: Fixation and Cell Staining
17. Wash cells twice with 100 µL 

SECTION: Fixation and Cell Staining
18. Stain cells with the DAPI + Phalloidin-647 mixture. Add 100 µLto each well and incubate at Room temperaturefor 20 min
SECTION: Fixation and Cell Staining
19. Wash cells twice with 100 µL 
SECTION: Fixation and Cell Staining
20. Maintain wells with200 µLfor 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
21. Using the microscope ImageXpress Micro XLS+ (Molecular Devices), check the template for the Corning 3603 plate and the filters for DAPI (nuclei), FITC (gelatin), TxRed (CMPTX) and Cy5 (phalloidin-647).
SECTION: Cell Imaging by Epifluorescence HTS
22. Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
SECTION: Cell Imaging by Epifluorescence HTS
23. Check the wells using the 4X objective.
SECTION: Cell Imaging by Epifluorescence HTS
24. Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
SECTION: Cell Imaging by Epifluorescence HTS
25. Acquire the plate. Export metadata for analysis.
SECTION: Cell Imaging by Epifluorescence HTS
26. For representative images, change to 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
27. On FIJI (ImageJ), import HTD files through BioFormats.
SECTION: Gelatin Degradation Quantification on FIJI
28. Images should already be scaled. If not, adjust scale based on the objective lens used for acquisition.
SECTION: Gelatin Degradation Quantification on FIJI
29. Set measurements to contain "Area", "Standard Deviation", "Shape Descriptor", "Mean grey value", "Perimeter" and "Display label".
SECTION: Gelatin Degradation Quantification on FIJI
30. Concatenate all stacks into a single hyperstack.
SECTION: Gelatin Degradation Quantification on FIJI
31. Split channels and select the FITC stack for analysis.
SECTION: Gelatin Degradation Quantification on FIJI
32. 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
33. To measure the degraded area, analyze particles with a range of "5-Infinity" and select "Summarize".
SECTION: Gelatin Degradation Quantification on FIJI
34. Save CSV file. The degraded area (in µm2) per site will be compared between groups in the statistical analysis.
SECTION: Cell Counting on FIJI
35.   

SECTION: Cell Counting on FIJI
36. Split channels and select the DAPI stack for counting.
SECTION: Cell Counting on FIJI
37. Set a threshold to contain nuclei. Create a new stack with the binary images.
SECTION: Cell Counting on FIJI
38. To count cells, analyze particles with a range of "10-infinity" and select "Summarize". 
SECTION: Cell Counting on FIJI
39. Save CSV file. 
SECTION: Cell Morphology Analysis on FIJI
41. Split channels and select the Cy5 stack for analysis.
SECTION: Cell Morphology Analysis on FIJI
40.   

SECTION: Cell Morphology Analysis on FIJI
42. Duplicate the stack as a guide.
SECTION: Cell Morphology Analysis on FIJI
44. Set a threshold to encompass cell cytoplasm. Create a new stack with the binary images.
SECTION: Cell Morphology Analysis on FIJI
47. Save CSV file. The cell circularity index of each cell will be compared between groups in the statistical analysis.
SECTION: Cell Morphology Analysis on FIJI
46. To analyze cell morphology, analyze particles with a range of "10-Infinity" and check "Clear Results".
SECTION: Cell Morphology Analysis on FIJI
45. 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. Merge the Cy5 stack with the TxRed stack to visually differentiate cell types.
SECTION: Image Processing for Representative Cells
48.   

SECTION: Image Processing for Representative Cells
49. Split the channels of the stacks of interest.
SECTION: Image Processing for Representative Cells
50. Adjust channel colors using the "Lookup Tables" menu.
SECTION: Image Processing for Representative Cells
51. Select the cell of interest in a 200x200 px squared selection.
SECTION: Image Processing for Representative Cells
52. Save selections in each channel and the combination of all channels in PNG images.