To confirm the specificity of chemical inhibitors in inhibiting MMP14 activity, gelatin degradation assay was performed using QCM™ Gelatin Invadopodia Assay (Millipore). Cy3-labelled gelatin matrix was first prepared according to manufactory’s instruction, then coated with an additional layer of FN. CNC explants were isolated as described above and seeded on the matrix. 0.2mM of NSC405020 or Prinomastat was added into the culture media before explants were plated. The explants were allowed to spread and migrate for 40 hours before imaged with 40x objective. The rate of gelatin degradation is quantitated by comparing the degraded areas that devoid of fluorescence to the total cell areas.
Qcm gelatin invadopodia assay
Manufactured by Merck Group
Sourced in United States
The QCM Gelatin Invadopodia Assay is a laboratory equipment product designed to measure the formation and activity of invadopodia, which are specialized cellular structures involved in extracellular matrix degradation. The assay utilizes quartz crystal microbalance (QCM) technology to quantify the degradation of a gelatin-coated surface, providing insights into the invasive potential of cells.
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Lab products found in correlation
Crystal violet, by SCP Science (2 mentions)
Alexa fluor 488 green, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 red, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Promega (2 mentions)
Rabbit anti cdc42, by Santa Cruz Biotechnology (2 mentions)
Rhoa rac1 cdc42 activation assay combo kit, by Cell Biolabs (2 mentions)
Rhodamine phalloidin stain, by Thermo Fisher Scientific (2 mentions)
Cell proliferation reagent, by Roche (2 mentions)
Primary mouse anti tks4, by Merck Group (2 mentions)
Primary mouse anti stard13, by Santa Cruz Biotechnology (2 mentions)
Dapi stain, by Roche (2 mentions)
Lsm 980 microscope, by Zeiss (1 mentions)
Tcs sp5, by Leica (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Su6656, by Merck Group (1 mentions)
Rk 20449, by Merck Group (1 mentions)
Gm6001, by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
15 protocols using qcm gelatin invadopodia assay
1
Quantifying MMP14 Inhibitor Activity
2
Quantitative Gelatin Invadopodia Assay
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Coverslips were inverted on 200 μL drop of QCM Gelatin Invadopodia Assay (Millipore) and heated to 37 °C. Coverslips were fixed in 0.5% glutaraldehyde for 15 min at 4 °C and after washing with PBS. Slides were sterilized with 70% ethanol and left in complete growth media for 1 h before use. Cells were cultured on gelatin-coated coverslips in a 24-well plate and left to adhere. Cells were then incubated for 48 hr in different experimental conditions and finally fixed and stained for CLSM examinations. Fluorescence signals were analyzed by Zeiss LSM980 Microscope equipped with a 63× oil objective. 3D reconstruction images of selected regions of interest (ROI) with evident matrix degradation spots were shown. To quantify invadopodia activity, black-and-white images of gelatin degradation are analyzed using FIJI software. The fraction of degraded area was analyzed in this way: Analyze>Set Measurements>select “Area Fraction”. The “area fraction” value was normalized to the number of nuclei in each image as measured from the DAPI channel in the same field [49 (link)].
3
Dissecting Cdc42 and RhoA Signaling
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Cdc42 and RhoA biosensors were kind gifts from Dr. Louis Hodgson (Albert Einstein College of Medicine). RhoA/Rac1/Cdc42 Activation Assay Combo Kit was purchased from Cell BioLabs (Sand Diego, CA, USA). QCM Gelatin Invadopodia Assay was obtained from Millipore (Massachusetts, USA). Primary antibodies against Cdc42, StarD13, actin, vinculin, Arp2, cortactin (Mouse and Rabbit) and TKS5 antibodies were obtained from Abcam (Cambridge, UK). Primary mouse anti-TKS4 was obtained from Merck Millipore. Primary mouse anti-StarD13 and rabbit anti-Cdc42 were obtained from Santa Cruz Biotech. HRP-conjugated secondary antibodies were obtained from Promega (Wisconsin, USA). Fluorescent secondary antibodies Alexa Fluor 488-green and Alexa Fluor 594-red as well as Rhodamine-phalloidin stain were purchased from Invitrogen (Massachusetts, USA). DAPI stain, and cell proliferation reagent were acquired from Roche Diagnostics (Roche Ltd, Mannheim, Germany). Hiperfect transfection reagent, luciferase and human Flexi Tubes siRNA for luciferase, StarD13, Cdc42 and RhoA were obtained from Qiagen (Hilden, Germany). Lipofectamine LTX was from Waltham (Massachusetts, USA) and crystal violet was from SCP Science (Quebec, Canada).
4
Rapid Invadopodia Visualization in HCC Cells
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QCM™ gelatin invadopodia assay (No.ECM671, Millipore, MA, USA) was used for rapid detection of matrix degradation [25 (link)]. In belief, 2 × 104/500 μl of HCC cells were added into glass dish pre-prepared with fluorescent substrate. Then the cells were cultured in the dark at room temperature for 15–30 min and sequentially in an incubator for 48 h. After fixed with 3.7% formaldehyde for 30 min, and stained with FITC-phalloidin and DAPI in blocking/permeabilization buffer for 1 h respectively, the cells were removed from staining solution and rinsed twice with fluorescent staining buffer using 500 μl/well. Laser scanning confocal microscope-TCS SP5 (Leica) was used to capture images.
5
Quantitative Analysis of Invadopodia Formation
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Invadopodia assay (QCM™ Gelatin Invadopodia Assay, Millipore) was performed according to the manufacturer’s instructions. Briefly, glass chamber slides were coated with Poly L Lysine, activated by a diluted glutaldehyde solution, and then fluorescently coated with fluorescent gelatin as the substrate for invadopodia. After disinfecting with 70% alcohol and quenching of free aldehydes with growth medium, cells were seeded onto the gelatin surface for 16 hours in complete media. Cells were then fixed in 4% formaldehyde in DPBS and visualized by nuclear (DAPI) and cytoskeleton staining (TRITC-Phalloidin) by fluorescent microscopy. Degraded area/puncta of fluorescent gelatin (devoid of green fluorescence) indicated the presence of invadopodia and was quantified per number of cells (at least 100 cells were analysed per experiment).
6
Dissecting Cdc42 and RhoA Signaling
7
Cell Invasion Assay with HA Mimetic
8
Invadopodia Degradation Assay in Hypoxia
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QCM Gelatin Invadopodia Assay (Millipore, MA, USA) performed as per manufacturer’s instructions in 8-well Lab-Tek® chamber slides (Nunc, IL, USA). Briefly, cells seeded at 1.6 × 104/well onto GFP-tagged gelatin to examine invadopodial matrix-degradation. After 72–96 h ± hypoxia, cells were fixed and stained with kit-supplied DAPI nucleic acid stain and filamentous actin-binding TRITC-phalloidin. Coverslips mounted with ProLong Gold Antifade Reagent (Invitrogen, OR, USA) and cells visualized using an Axioskop 40 epifluorescent microscope (20× objective) (Zeiss, Göttingen, Germany). Five fields of view obtained per condition. Gelatin degradation, cell area and cell counts quantified using ImageJ (V1.46; NIH).
9
Quantifying Astrocyte Invadopodia Formation
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Human primary astrocytes incubated with and without GBM-derived EVs were analyzed using the QCM Gelatin Invadopodia Assay (Millipore). Astrocytes were seeded at 14,000 cells/cm2 in growth medium on the Cy3-gelatin surface and incubated with 10-μg GBM-derived EVs (1 μg EV per 1120 cells; 12.5 μg per cm2) for 24 h at 37 °C in 5% CO2 in triplicate. Cells were fixed and stained with FITC-phalloidin (1:100) and DAPI (1:200) as previously described [24 (link)]. Samples were visualized on an Olympus BX51 fluorescence microscope at ×10 objective magnification for quantification. Image analysis was performed using ImageJ (National Institutes of Health, USA) [29 (link)]. A high-intensity threshold was set for positive DAPI signal, and then analyzed as “particles” for cell counting. Similarly, a high-intensity threshold for the phalloidin signal enabled measurement of the total cell area. A low-intensity threshold was set for areas devoid of fluorescent gelatin to enable quantification of total gelatin degradation.
10
Measuring Macrophage Gelatin Degradation
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