The motility of the cells was also evaluated using cell culture inserts from BD Falcon (Franklin Lakes, NJ), and following the protocol previously established with minor modifications [44] (link). Cells that were previously maintained in a starvation medium with 0.2% FBS for 24 hr to minimize cellular proliferation, were resuspended at a concentration of 2×105 cells/ml in medium containing 0.2% FBS. 1×105 cells or 500 µl of cell suspensions were plated onto each insert, which was previously coated with 3 µg/ml of rat tail collagen solution from BD (Bedford, MA) overnight at room temperature. The lower chamber contained medium with 10% FBS as a chemo-attractant. Cells were allowed to pass through the porous membrane and were collected at different time points that were empirically determined based on the migratory ability of each cell line. Non-migratory cells were then removed from the surface of the membranes using cotton swabs. Cells that passed through the pores of the membrane were fixed and stained using the Diff-Quick nuclei and cytoplasm staining kit (Dade Behring, Newark, DE). Stained migratory cells were counted under a microscope. Each experiment was performed in triplicates and was repeated at least once to validate the initial data.
Rat tail collagen solution
Manufactured by BD
Sourced in Morocco
Rat tail collagen solution is a laboratory product that consists of a soluble form of collagen extracted from the tails of rats. This solution is a common component used in various cell culture and tissue engineering applications.
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Lab products found in correlation
5 protocols using rat tail collagen solution
1
Cell Migration Assay Protocol
2
3D Culture Model of Ovarian Cancer Cells
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Rat tail collagen solution (BD Biosciences, San Jose, CA) at 3.4 mg mL−1 was diluted with 0.1% HAc (v/v) to maintain the final concentration at 2 mg mL−1 for all experiments. Before casting the 3D cultures in cell culture plates, the bottom of each well was pre-coated with the diluted collagen solution at 37 °C for 2 h. To generate the 3D scaffold, OV-NC and OV-206 cells were trypsinized and resuspended as individual cells at 2 × 106 cells per mL. The cell suspension was mixed with 2 mg mL−1 collagen hydrogel and 10 × DMEM, using 0.1% (v/v) NaOH to adjust to neutral pH. According to the previous preparation techniques of our laboratory, we constructed a 3D culture model of OvCa cells. The final cell concentration in the model was 2.5 × 105 cells per mL. Once in plate, the collagen gel solution was kept in a humidity box at 37 °C for 2 h to allow gel polymerization via thermal cross-linking; then, 1.5 mL fresh cell culture medium was gently added to each well. The medium was refreshed every other day.
3
Collagen Gel Contraction Assay
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MLF were trypsinized and seeded in a 4 mg/mL rat-tail collagen solution (BD Biosciences, San Jose, CA) at a density of 3 x 105 per mL in a 12-well plate. Gels were allowed to solidify, then released from the plate and allowed to contract in serum-free medium. Reduction of gel diameter was calculated in each condition using ImageJ.
4
Measuring Cell Invasiveness via Transwell Assay
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The invasiveness of the cells was measured using cell culture inserts from BD Falcon (Franklin Lakes, NJ) by following a previously described protocol with minor modifications [45] (link). As in the transwell-based motility assay described above, cells were maintained in a starvation medium with 0.2% FBS for 24 hr before seeding to minimize cellular proliferation. Inserts were coated with 50 µg/ml of rat tail collagen solution from BD (Bedford, MA) for 5 hr at room temperature. After the incubation, inserts were washed three times with serum-free medium and were allowed to dry at 37°C overnight. Once dried, membranes were covered with 100 µl of collagen solution at a final concentration of 1.3 mg/ml (for all cells) or with 100 µl of Matrigel (Cat. #356231) from BD (Bedford, MA) at a final concentration of 300 µg/ml (for Myc-CaP cells only), and were allowed to solidify at 37°C. Cells were resuspended at a concentration of 2×105 cells/ml in medium containing 0.2% FBS. 1×105 cells or 500 µl of cell suspensions were plated onto each insert. The remaining procedure was performed as described above in the motility assay section. Each experiment was performed in triplicates and was repeated at least once to validate the initial data.
5
Multicellular Tumor Spheroid Invasion Assay
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Multicellular tumor spheroids were generated by using the hanging-drop method39 (link). In brief, CRT-MG/IL-8p-dEGFP cells were detached with EDTA (2 mM), resuspended in RPMI1640 with 10% FBS, 20% methylcellulose (Sigma-Aldrich, Co.), and 1% Matrigel (BD Biosciences, San Jose, California), and incubated as droplets (25 μl) containing 1,000 cells for 48 h to ensure multicellular aggregation. For collagen invasion assays, spheroids were mixed with rat-tail collagen solution (3 mg/ml, BD Biosciences), pipetted as a drop-matrix, and then polymerized at 37 °C. Necrotic cells were added to the spheroid cultures 24 h after seeding. The invasion area was measured by using Image J software as previously described with minor modification40 (link). In brief, invading area was calculated as follows: total area minus spheroid body area. These value were analyzed in seven independent spheroids per condition. The experiment was performed in triplicates.
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