The protocol for CDM growth was adapted from existing methods (Kaukonen et al., 2017 (link)), (Castelló-Cros and Cukierman, 2009 (link)). First, microscope coverslips (Ø 18 mm, Superior Marienfeld) were cleaned with soap and water, dried with N2, and disinfected with ethanol and UV. Then, they were incubated with 1% sterile gelatin (from porcine skin, Sigma-Aldrich) in PBS and cross-linked with 1% filtered glutaraldehyde in PBS (Sigma-Aldrich). The reaction was quenched with 1 M sterile glycine (Sigma-Aldrich) solution in PBS. Gelatin coatings of a thickness of 1 ± 0.5 µm were obtained, for which an elastic modulus between 0.1-0.2 MPa has been reported, thus matching the elastic modulus range of human skin (Kuo et al., 2021 (link)), (Wei et al., 2017 (link)). The coated coverslips were used immediately or stored in the fridge with 1% Penicillin and Streptomycin in PBS for a maximum of two weeks. To assemble the matrices, 1 ml of medium containing 5 × 104 cells/cm2 of primary fibroblasts were added on the gelatin-coated substrate. After one day, if confluency was achieved, ascorbic acid (AA) (Sigma-Aldrich) treatment was started. Standard culture media supplemented with 50 μg/ml AA was added every two days for a maximum of eight days to stimulate the generation of collagen and stabilize the generated matrix.
Sterile gelatin
Manufactured by Merck Group
Sterile gelatin is a purified and sterilized form of gelatin, a protein derived from collagen. It is a colorless, odorless, and tasteless material that solidifies when cooled and liquefies when heated. Sterile gelatin is commonly used as a gelling agent in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e200 microscope, by Nikon (4 mentions)
Multiskan ex, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Microscope coverslips, by Marienfeld (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
40 6 diamidino 2 phenylindole dapi, by Merck Group (1 mentions)
Goat anti mouse igg alexa fluor 488 a 11001, by Thermo Fisher Scientific (1 mentions)
Fv 10 asw 1, by Olympus (1 mentions)
Texas red phalloidin, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Endothelial cell growth supplement from bovine pituitary, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Endothelial cell growth supplement (ecgs), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
7 protocols using sterile gelatin
1
Collagen-Derived Matrix (CDM) Growth Protocol
2
Immunofluorescence Imaging of FAK and HER2
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SKBR3 and BT-474 cells were grown on coverslips previously coated with 1% sterile gelatin (Sigma-Aldrich) and exposed for 72 h to 1-10 μg/ml Tz, 10-6M RA or the combination of both drugs. Cells were fixed with 4% paraformaldehyde for 35 min and permeabilized with 0.1% Triton for 5 min. Blocking step was performed with 0.5% bovine serum albumin solution for 30 min at room temperature. Cells were incubated with the first antibody against FAK (Mouse, clone 77, BD Biosciences) and against HER2 (Mouse, catalogue 16901-Abcam) overnight at 4°C. After washing, cells were incubated with Goat Anti-Mouse IgG-Alexa Fluor 488 (A-11001, Invitrogen) for 90 min at room temperature. The cells were washed and then stained for 35 min with Texas Red phalloidin (Sigma-Aldrich) to reveal actin and the nuclei counter stained with 40-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. The coverslip cells were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were captured by using a Nikon Eclipse E200 microscope (Tokyo, Japan) coupled to a high-resolution 590CU 5.0M CCD digital camera or examined under fluorescence microscopy (FV1000 Olympus Confocal Microscope) and the FV 10-ASW 1.7 software (Olympus, Japan).
3
Regulation of Cell Adhesion by RA and FAKi
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Cells were exposed to RA (1–10 μM), FAKi (1 μM) or their combination RA (1 μM) plus FAKi (1 μM) for 72 h. Fifteen thousand T-47D cells/well, fifty thousand LM3 cells/well, and twenty thousand HeLa cells/well were seeded into 96-well plates previously coated with 1% sterile gelatin (Sigma-Aldrich). Cells were incubated at 37 °C for 2 h. Non-adherent T-47D cells were removed by gentle washing with PBS. The attached cells were fixed with 4% paraformaldehyde and stained with 10% ethanol/crystal violet for 20 min. Images were captured and counted in ten randomly chosen fields per well using a Nikon Eclipse E200 microscope coupled to a high-resolution CCD digital camera (Nikon, Tokyo, Japan), as previously described [26 (link)]. Cell adhesion was calculated as a percentage of attached treated cells compared to untreated cells.
4
Adherent Cell Assay with Tz and RA
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Cells were exposed to 1-10 μg/ml Tz, 10-6M RA or the combination of both drugs for 72 h. Then, 10,000 SKBR3 cells/well or 50,000 BT-474 cells/well were seeded into 96-well plates previously coated with 1% sterile gelatin (Sigma-Aldrich). The cells were incubated at 37°C in a tissue culture incubator. The culture medium was removed after 1 h and washed with PBS to remove any non-adherent cells. The attached cells were fixed/stained with 10% ethanol/crystal violet for 20 min. Absorbance at 570 nm was measured with a microplate reader (MULTISKAN EX; Thermo Scientific, Lafayette, CO, USA) and images of attached cells were captured by a Nikon Eclipse E200 microscope coupled to a high-resolution 590CU 5.0M CCD digital camera.
5
Quantifying Cell Adhesion Dynamics
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Five hundred thousand cells per well were seeded into 6-well plates on coverslips previously coated with 1% sterile gelatin (Sigma-Aldrich) and exposed to different treatments. The cells were incubated at 37°C (in tissue culture incubator) and after 1 hr, the plates were shaken for 1 min. at 150 r.p.m. and washed with PBS to remove any non-adherent cells. The attached cells were fixed with 4% formaldehyde and stained with Giemsa. Cells attached images were captured and counted by using a Nikon Eclipse E200 microscope coupled to a high-resolution 590CU 5.0M CCD digital camera.
6
Isolation and Culture of HUVECs
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HUVECs were isolated from fresh umbilical cords using mild collagenase (Sigma-Aldrich, St Louis, MO) treatment and by flushing with Hanks' Balanced Salt Solution (HBSS; Sigma-Aldrich) as described previously. 13 , 14 HUVECs were grown in M199 Medium (Gibco; Invitrogen, Carlsbad CA) containing 20% fetal calf serum (Biochrom, Berlin, Germany), 5 μg/mL endothelial cell growth supplement from bovine pituitary (Sigma-Aldrich), 50 U/mL heparin, and 1% penicillin/streptomycin (Gibco; Thermo Fisher, Waltham, MA). Cells were seeded into 6-well plates (Costar; Corning Incorporated, Corning, NY) precoated with 1% sterile gelatin (Sigma-Aldrich) at a density of 5 × 10 5 cells/plate and cultured under standard conditions. After serum starvation in M199 containing 0.1% bovine serum albumin and endothelial cell growth supplement (ECGS) for 16 hours (Sigma-Aldrich), drug exposures were performed. Proteasome inhibition was induced by pretreatment of HUVECs with 1 μM lactacystin for 30 minutes.
Cell preparations were obtained from different donors and repeated 3 times, so as to obtain 3 different "donor cell pools."
Cell preparations were obtained from different donors and repeated 3 times, so as to obtain 3 different "donor cell pools."
7
Adherence Assay for Anti-Cancer Drugs
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Cells were exposed for 72 h to the mono drugs (1 μg/mL Tz, 1μg/mL Pz, 1 μg/mL T-DM1, 0.1 μg/mL Lp) and the combinations (1:0.1 μg/mL Tz + Lp, 1:0.1 μg/mL T-DM1 + Lp, 1:1 μg/mL Tz + Pz) with and without the transfection with specific siRNAs versus FAK, paxillin, and cortactin. After the treatments, cells were trypsinized and suspended in PBS containing trypan blue to determine the number of viable cells and seed the same viable cell density for each experimental condition. Briefly, 3 × 104 SKBR3, 5 × 104 BT-474, and 3 × 104 SKBR3-RTz cells/well were seeded into 96-well plates previously coated with 1% sterile gelatin (Sigma-Aldrich). Cells were incubated at 37 °C for 2 h. Non-adherent cells were removed by gentle washing with PBS. The attached cells were fixed with 4% p-formaldehyde and stained with 10% ethanol/crystal violet for 20 min. Absorbance at 570 nm was measured using a microplate reader (MULTISKAN EX; Thermo Scientific, St. Leon-Rot, Germany). The absorbance is directly proportional to the number of adherent cells. The results are expressed as the percentage of cell adhesion, normalized with respect to control, which is considered 100%. Images were captured using a Nikon Eclipse E200 microscope coupled with a high-resolution CCD digital camera. The protocol was previously described in Castro-Guijarro et al. (2022) [36 (link)].
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