TITLE: Indirect Co-Culture Assay using Boyden Chambers
AUTHORS: Bianca Cruz Pachane, Heloisa Sobreiro Selistre de Araujo

[DESCRIPTION]
Here, we describe a transwell assay using Boyden chambers as an indirect co-culture method, where two cell lines are grown together but separated by a porous membrane. This assay was used to determine the effect of hypoxic, tumoral EVs in cells from the tumor microenvironment without direct contact between them, focusing on intercellular adhesion and tumoral invasion.

[BEFORE_START]
Fluorescent gelatin preparation: Under sterile conditions, solubilize the fluorescent gelatin stock at 37 °Cwith 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 °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: Preparation of Gelatin-Coated Coverslips
1. In preparation: 
Clean round glass coverslips (13 mm ø) with 70% ethanol wipes before use. Maintain slips in a clean container.
Prepare a 0.5 % (v/v)and keep at 4 °C protected from light.

SECTION: Preparation of Gelatin-Coated Coverslips
3. Drop coverslips atop the droplets and incubate at Room temperature for 20 min .
SECTION: Preparation of Gelatin-Coated Coverslips
2. Under sterile conditions, apply 20 µL in spaced droplets to a surface covered in   

SECTION: Preparation of Gelatin-Coated Coverslips
4. Using forceps, transfer the coverslip to a 24-well plate with the coating facing upwards. 
SECTION: Preparation of Gelatin-Coated Coverslips
5. Wash coverslips twice with 500 µL 

SECTION: Preparation of Gelatin-Coated Coverslips
6. Cross-link coating with 500 µLof cold  0.5 % (v/v) for15 min at Room temperature 

SECTION: Preparation of Gelatin-Coated Coverslips
7. Prepare a Petri dish with the bottom covered in   

SECTION: Preparation of Gelatin-Coated Coverslips
8. Apply spaced 20 µL droplets of the fluorescent gelatin working solution at 0.2 mg/mL to the Parafilm-covered surface.
SECTION: Preparation of Gelatin-Coated Coverslips
9. Remove the coverslips from the 24-well plate and drop them atop the droplets, with the coating facing down. Incubate at 4 °C  20 min , protected from light.
SECTION: Preparation of Gelatin-Coated Coverslips
10. The next day, remove the slips from the Petri dish using a forceps and transfer them, with the coating facing up, to a fresh 24-well plate.
SECTION: Preparation of Gelatin-Coated Coverslips
11. Wash coverslips thrice with 500 µL 

SECTION: Preparation of Gelatin-Coated Coverslips
11.1. Slips can be stored at 4 ºC for up to a week, wrapped in aluminium foil.
SECTION: Preparation of Gelatin-Coated Coverslips
12. Pre-condition gelatin coating with 500 µL for 30 min at 37 °C  without CO2.

SECTION: MDA-MB-231 Seeding in Gelatin-Covered Coverslips
13. Subculture cells as usual. Resuspend cell pellets in Leibovitz L-15 10% FBS and count cells using the trypan blue exclusion method.
SECTION: MDA-MB-231 Seeding in Gelatin-Covered Coverslips
15. Seed 5x104 cells in 1000 µL in each well. Seal the plate with   
SECTION: MDA-MB-231 Seeding in Gelatin-Covered Coverslips
14. Remove the pre-conditioning medium from the 24-well plate.
SECTION: MDA-MB-231 Seeding in Gelatin-Covered Coverslips
16. Incubate cells at 37 °C without CO2   for adhesion.

SECTION: HUVEC Seeding in Transwell Insert
17. Subculture cells as usual. Resuspend cell pellets in DMEM 10% FBS 1% pen/strep and count cells using the trypan blue exclusion method.
SECTION: HUVEC Seeding in Transwell Insert
18. Add 750 µLto the wells of a 24-well plate (Corning).
SECTION: HUVEC Seeding in Transwell Insert
19. Assemble a Boyden chamber using a 24-well plate and 3.0 µm inserts (Falcon).
SECTION: HUVEC Seeding in Transwell Insert
20. Seed 5x104 cells in 250 µL to the upper chamber.

SECTION: HUVEC Seeding in Transwell Insert
21. Incubate cells at 37 °C  for adhesion.

SECTION: HDFa Seeding in Transwell Insert
22. Subculture cells as usual. Resuspend cell pellets in DMEM 10% FBS 1% pen/strep and count cells using the trypan blue exclusion method.
SECTION: HDFa Seeding in Transwell Insert
23. Add 750 µLto the wells of a 24-well plate (Corning).
SECTION: HDFa Seeding in Transwell Insert
24. Assemble a Boyden chamber using a 24-well plate and 8.0 µm inserts (Falcon).
SECTION: HDFa Seeding in Transwell Insert
25. Seed 2x103 cells in 250 µL to the upper chamber.

SECTION: HDFa Seeding in Transwell Insert
26. Incubate cells at 37 °C  for adhesion.

SECTION: Indirect Co-Culture Assembly
27. Remove the growth media from all wells and Boyden chambers (both the upper and the lower chambers).
SECTION: Indirect Co-Culture Assembly
28. Transfer the MDA-MB-231 coverslips to a fresh well.
SECTION: Indirect Co-Culture Assembly
29. Add 750 µL to the coverslip-containing wells.

SECTION: Indirect Co-Culture Assembly
30. Transfer either the HUVEC or the HDFa inserts to the wells.
SECTION: Indirect Co-Culture Assembly
31. Add 250 µL containing EVh (109 particles/ml) or the equivalent treatment volume in PBS to the upper chamber.

SECTION: Indirect Co-Culture Assembly
32. Incubate cells at 37 °C for 1440 min 

SECTION: Fixation and Cell Staining
33. Collect both the upper and the lower chamber's supernatant into a 1.5 ml microtube. Keep on ice . This will be processed later for gelatin zymography -   

SECTION: Fixation and Cell Staining
34. Transfer the inserts to new wells.
SECTION: Fixation and Cell Staining
35. Fix cells with 1000 µL for 10 min at Room temperature 

SECTION: Fixation and Cell Staining
36. Wash wells twice with 500 µL 

SECTION: Fixation and Cell Staining
38. Permeabilize cells with 100 µL 0.1% Triton X-100 at Room temperaturefor 5 min

SECTION: Fixation and Cell Staining
39. Wash wells twice with 500 µL 

SECTION: Fixation and Cell Staining
41. Wash wells twice with 500 µL 

SECTION: Fixation and Cell Staining
40. Stain cells with the DAPI + Phalloidin-647 mixture. Add 20 µL of staining solution in spaced droplets to a Parafilm-covered surface. Drop coverslips (coating facing down) and insert membranes atop the droplets and incubate at Room temperature, protected from light for 20 min

SECTION: Fixation and Cell Staining
37. Clean the outside part of the insert membrane with a swab to remove any debris.
SECTION: Fixation and Cell Staining
42. Assemble coverslips to clean histological slides using Fluoromount as mounting media. Allow slide to dry for at least 240 min, protected from light.

SECTION: Fixation and Cell Staining
43. Detach membranes from inserts using a scalpel. Assemble the membranes in histological slides using Fluoromount as mounting media. Seal the membrane with a rectangular coverslip and allow slide to dry for at least 240 min , protected from light.

SECTION: Fixation and Cell Staining
44. Once dry, seal coverslips using clear nail polish and store at 4 °C 

SECTION: Cell Imaging by Epifluorescence HTS
45. Using the microscope ImageXpress Micro XLS+ (Molecular Devices), check the template for the histological slides and the filters for DAPI (nuclei), FITC (gelatin - for MDA-MB-231 only) and Cy5 (phalloidin-647).
SECTION: Cell Imaging by Epifluorescence HTS
46. Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
SECTION: Cell Imaging by Epifluorescence HTS
47. Check the wells using the 4X objective.
SECTION: Cell Imaging by Epifluorescence HTS
48. Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
SECTION: Cell Imaging by Epifluorescence HTS
49. Acquire the plate. Export metadata for analysis.
SECTION: Cell Imaging by Epifluorescence HTS
50. 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
51. On FIJI (ImageJ), import HTD files through BioFormats.
SECTION: Gelatin Degradation Quantification on FIJI
52. Images should already be scaled. If not, adjust scale based on the objective lens used for acquisition.

SECTION: Gelatin Degradation Quantification on FIJI
53. Set measurements to contain "Area", "Standard Deviation", "Shape Descriptor", "Mean grey value", "Perimeter" and "Display label".
SECTION: Gelatin Degradation Quantification on FIJI
54. Concatenate all stacks into a single hyperstack.
SECTION: Gelatin Degradation Quantification on FIJI
55. Split channels and select the FITC stack for analysis.
SECTION: Gelatin Degradation Quantification on FIJI
56. 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
57. To measure the degraded area, analyze particles with a range of "5-Infinity" and select "Summarize".
SECTION: Gelatin Degradation Quantification on FIJI
58. Save CSV file. The degraded area (in µm2) per site will be compared between groups in the statistical analysis.
SECTION: Cell Morphology Analysis on FIJI
59.   

SECTION: Cell Morphology Analysis on FIJI
60. Split channels and select the Cy5 stack for analysis.
SECTION: Cell Morphology Analysis on FIJI
62. Set a threshold to encompass cell cytoplasm. Create a new stack with the binary images.
SECTION: Cell Morphology Analysis on FIJI
63. Using the duplicated stack as a guide, section cells using the "pencil" tool with a 3 px thickness.
SECTION: Cell Morphology Analysis on FIJI
64. To analyze cell morphology, analyze particles with a range of "10-Infinity" and check "Clear Results".
SECTION: Cell Morphology Analysis on FIJI
65. Save CSV file. The cell circularity index of each cell will be compared between groups in the statistical analysis.
SECTION: Gelatin Zymography of Assay Supernatant
67. Spin the assay conditioned media at 1200 rpm, 10 min, 4 °C. Transfer the supernatant to new tubes and discard pellet.
SECTION: Gelatin Zymography of Assay Supernatant
68. Quantify the supernatants using the Pierce™ BCA Protein Assay kit, following the manufacturer's instructions. A plate reader is required for this step.
SECTION: Gelatin Zymography of Assay Supernatant
67.1. Store microtubes at -80 °C until the time of analysis.
Thaw microtubes on ice  before the next step and avoid freeze/thawing cycles.

SECTION: Gelatin Zymography of Assay Supernatant
69. Aliquot samples in microtubes to contain 15 µg per well.
SECTION: Gelatin Zymography of Assay Supernatant
70. Mix samples with non-reducing Laemmli sample buffer (10 % (w/v); glycerol; 0.1 Molarity (M); 1 Molarity (M); bromophenol blue) at a 1:2 ratio. 

SECTION: Gelatin Zymography of Assay Supernatant
66. Prepare 10% SDS-PAGE gels containing gelatin (100 mg/ml) using the Mini-PROTEAN system (Bio-Rad) with 0.75 mm spacers. Maintain gels hydrated at 4 °C    after polymerization.
SECTION: Gelatin Zymography of Assay Supernatant
71. Assemble gels in running buffer and load samples onto gels alongside a protein ladder (Precision Plus Protein™ Dual Color, Bio-Rad).
SECTION: Gelatin Zymography of Assay Supernatant
72. Run electrophoresis at 80 V at 4 °C for circa 240 min .
SECTION: Gelatin Zymography of Assay Supernatant
73. Transfer gels onto cases and photograph them using the ChemiDOC Imaging System (Bio-Rad).
SECTION: Gelatin Zymography of Assay Supernatant
74. Wash gels with 2.5 % (v/v) for 40 min under orbital agitation.
SECTION: Gelatin Zymography of Assay Supernatant
75. Incubate gels in buffer (50 millimolar (mM) 5 millimolar (mM) 0.02 % (w/v) 10 millimolar (mM) ) at 37 °C for 1200 min 

SECTION: Cell Morphology Analysis on FIJI
61. Duplicate Cy5 stack.
SECTION: Gelatin Zymography of Assay Supernatant
76. Stain gels with Coomassie Brilliant Blue for 1440 min under agitation at Room temperature .

SECTION: Gelatin Zymography of Assay Supernatant
77. Destain gels with acetic acid:methanol:water (1:1:8, v/v) for up to 2880 min 
SECTION: Gelatin Zymography of Assay Supernatant
78. Photograph gels using the ChemiDOC Imaging System (Bio-Rad).
SECTION: Gelatin Zymography of Assay Supernatant
79. Perform a band densitometry analysis in each gel.