Sep 09, 2024
Fluorescent Gelatin Degradation Assay to Evaluate EVh Action in TME Cells
- Bianca Cruz Pachane1,
- Heloisa Sobreiro Selistre de Araujo1
- 1Department of Physiological Sciences, Universidade Federal de São Carlos - UFSCar
Protocol Citation: Bianca Cruz Pachane, Heloisa Sobreiro Selistre de Araujo 2024. Fluorescent Gelatin Degradation Assay to Evaluate EVh Action in TME Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn615ml5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 07, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 107099
Keywords: Cell Invasion, Gelatin Degradation, Gelatinase
Funders Acknowledgement:
São Paulo Research Foundation
Grant ID: 2021/01983-4
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Abstract
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).
Materials
Materials and reagents
- Corning 96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates, Individually Wrapped
- Sterile microtubes and pipettes
- Gelatin From Pig Skin, Fluorescein Conjugate, Thermo Fisher - Catalog #G13187
- Sterile PBS
- OptiMEM I Reduced Serum Media, Gibco - Catalog #31985070
- Trypan Blue solution 0.4%, Merck Millipore (Sigma-Aldrich) - Catalog #T8154-100 ml
- Paraformaldehyde solution (PFA 4% in deionized water, pH 7.6 - Sterile)
- Triton X-100 0.1% (v/v) in deionized water
- Phalloidin + DAPI (1 µl Phalloidin-iFluor 647, Abcam - Catalog #ab176759 + 0.76 µL 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI), Thermo Fisher Scientific - Catalog #D1306 in 5 mL PBS)
Cell lines and growth media
- MDA-MB-231 (ATCC® CRM-HTB-26™) - Leibovitz L-15 10% FBS
- HDFa (ATCC® PCS-201-012™) - DMEM 10% FBS 1% pen/strep
- HUVEC (ATCC® CRL-1730™) - DMEM 10% FBS 1% pen/strep
- THP-1 (ATCC® TIB-202™) - RPMI 1640 10% FBS 1% pen/strep
Equipments:
- Biological cabinet
- Cell incubator (37 ºC, 5% CO2)
- Cell counter - TC20 Cell Counter, Bio-Rad - Catalog #1450011
- Epifluorescence microscope - ImageXpress Micro XLS, Molecular Devices - Catalog #500496
Protocol materials
Parafilm
Step 18
Safety warnings
Light-sensitive assay. Work under sterile conditions.
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 00:30:00 . 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.
Fluorescent Gelatin Coating
Fluorescent Gelatin Coating
30m
30m
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.
Experimental plate map:
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | MDA-MB-231 PBS | MDA-MB-231 PBS | MDA-MB-231 PBS | MDA-MB-231 EVh | MDA-MB-231 EVh | MDA-MB-231 EVh |
| B | HUVEC PBS | HUVEC PBS | HUVEC PBS | HUVEC EVh | HUVEC EVh | HUVEC EVh |
| C | HDFa PBS | HDFa PBS | HDFa PBS | HDFa EVh | HDFa EVh | HDFa EVh |
| D | THP-1 PBS | THP-1 PBS | THP-1 PBS | THP-1 EVh | THP-1 EVh | THP-1 EVh |
| E | ||||||
| F | ||||||
| G | ||||||
| H |
| 7 | 8 | 9 | 10 | 11 | 12 | |
| A | ||||||
| B | ||||||
| C | ||||||
| D | ||||||
| E | ||||||
| F | ||||||
| G | ||||||
| H |
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.
Incubate plate for 00:30:00 at 37 °C 5% CO2 .
30m
Carefully remove excess coating (avoid touching well bottom).
Pre-condition coating with 200 µL OptiMEM for 00:30:00 at 37 °C 5% CO2 .
30m
Cell seeding
Cell seeding
1d
1d
Subculture cells as usual. Resuspend cell pellet in OptiMEM and count cells using the trypan blue exclusion method.
Remove pre-conditioning media from the wells (avoid touching well bottom).
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)
Treat cells with EVh (109 particles/ml) or the equivalent treatment volume in PBS.
Incubate plate for 24:00:00 at 37 °C 5% CO2
1d
Fixation and Cell Staining
Fixation and Cell Staining
10m
10m
Remove the supernatant by aspiration.
Fix cells with 100 µL warmed 4% PFA at Room temperature for 00:10:00
10m
Wash wells twice with 100 µL PBS
Permeabilize cells with 100 µL 0.1% Triton X-100 at Room temperature for 00:05:00
5m
Wash wells twice with 100 µL PBS
Stain cells with the DAPI + Phalloidin-647 mixture. Add 100 µL of staining solution to each well and incubate at Room temperature , protected from light for 00:20:00 .
20m
Wash wells twice with 100 µL PBS
Maintain wells with 200 µL PBS for analysis. Seal the plate with ParafilmContributed by users and cover it with aluminum foil for storage at 4 °C for up to 6 months.
Cell Imaging by Epifluorescence HTS
Cell Imaging by Epifluorescence HTS
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).
Set laser intensity to a minimum of 10 ms and increase gradatively if necessary.
Check the wells using the 4X objective.
Change into the 20x objective and adjust the laser focus. Select 9 sites per well minimally.
Acquire the plate. Export metadata for analysis.
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.
Gelatin Degradation Quantification on FIJI
Gelatin Degradation Quantification on FIJI
On FIJI (ImageJ), import HTD files through BioFormats.
Images should already be scaled. If not, adjust scale based on the objective lens used for acquisition.
Set measurements to contain "Area", "Standard Deviation", "Shape Descriptor", "Mean grey value", "Perimeter" and "Display label".
Concatenate all stacks into a single hyperstack.
Split channels and select the FITC stack for analysis.
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.
To measure the degraded area, analyze particles with a range of "5-Infinity" and select "Summarize".
Save CSV file. The degraded area (in µm2) per site will be compared between groups in the statistical analysis.
Cell Counting on FIJI
Cell Counting on FIJI
go to step #25 and follow through step #27
Split channels and select the DAPI stack for counting.
Set a threshold to contain nuclei. Create a new stack with the binary images.
To count cells, analyze particles with a range of "10-infinity" and select "Summarize".
Save CSV file.
Cell Morphology Analysis on FIJI
Cell Morphology Analysis on FIJI
go to step #25 and follow through step #27
Split channels and select the Cy5 stack for analysis.
Duplicate the stack as a guide.
Set a threshold to encompass cell cytoplasm. Create a new stack with the binary images.
Using the duplicated stack as a guide, section cells using the "pencil" tool with a 3 px thickness.
To analyze cell morphology, analyze particles with a range of "10-Infinity" and check "Clear Results".
Save CSV file. The cell circularity index of each cell will be compared between groups in the statistical analysis.
Image Processing for Representative Cells
Image Processing for Representative Cells
go to step #25 and follow through step #27
Split the channels of the stacks of interest.
Adjust channel colors using the "Lookup Tables" menu.
Select the cell of interest in a 200x200 px squared selection.
Save selections in each channel and the combination of all channels in PNG images.
Protocol references
PACHANE, Bianca Cruz et al. Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion. International Journal of Molecular Sciences, [s. l.], v. 23, n. 20, p. 12646, 2022.
EVEN-RAM, Sharona; ARTYM, Vira. Extracellular Matrix Protocols: Second Edition. [S. l.]: Humana Press, 2009.