MDA‐MB‐231 and MCF‐7 cell lines were trypsinized and suspended using trypsin (Gibco, pro. no. 25200‐56), and they were then centrifuged (100g for 5 min at room temperature). The suspended cells were counted using a hemocytometer lam (Marienfeld), and several droplets of 20 µl suspended cells (~1 × 103 cells) were deposited on the lid of a cell culture dish. The lid was returned to the cell culture dish (SPL Life Sciences), and the droplet hanged upside down and incubated for 2 days in a standard cell culturing incubator (37℃, 5% CO2, RH ~95%). Following that, the desired spheroids were collected and added to the collagen matrix (~100–150 µm, using an inverted microscope; grown spheroids should have a tight and compact spherical shape; disintegrated spheroids and those with loose aggregations are not suitable) (type I rat tail, Corning). The spheroid and collagen mixtures were incubated for 30 min (37℃, 5% CO2, RH ~95%). After gel cross‐linking, the cell culture medium was added to the combination.18 , 19
Type 1 rat tail
Manufactured by Corning
The Type I rat tail is a laboratory equipment product designed for specialized research applications. It serves the core function of providing a controlled and standardized sample for specific experimental protocols. The detailed specifications and intended use of this product are not included in this response.
Automatically generated - may contain errors
Lab products found in correlation
Cell culture dish, by SPL Life Sciences (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hibernate a medium, by Thermo Fisher Scientific (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Merck Group (1 mentions)
William s e medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
A1 confocal system, by Nikon (1 mentions)
5 protocols using type 1 rat tail
1
3D Spheroid Formation and Encapsulation Assay
2
Isolation and Culture of hMPCs
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Skeletal muscle tissue and primary hMPCs were obtained using previously published methods (Riddle et al., 2018b (link)). Briefly, SkM tissue was taken from the vastus lateralis using the percutaneous biopsy technique. Tissue was snap-frozen in liquid nitrogen and stored at -80°C (SkM) or stored in Hibernate®-A medium (Invitrogen) at 4°C until tissue disassociation. hMPCs were isolated as previously described (Xu et al., 2015 (link); Riddle et al., 2018b (link)) and were verified to be positive for the MPC specific transcription factor MyoD (Supplementary Figure S1 ). Passage six hMPCs were cultured in a 5% CO2 atmosphere at 37°C on collagen (Type I, Rat Tail, Corning) coated plates. Only female hMPCs were used due to sample availability.
3
Hepatocyte Isolation and Culture Protocol
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Hepatocytes were isolated by a two-step collagenase perfusion of livers as previously described (Rooney et al., 1989 (link); Thomas et al., 1991 (link)). Cell viability was determined by Trypan blue exclusion and typically ranged between 85 and 95%. Hepatocytes (7 × 105) were plated on collagen-coated (type I rat tail, Corning; 10 μg/cm2) glass coverslips in William’s E Medium (Thermo Fisher Scientific) supplemented, 2 mM glutamine, 10 units/ml penicillin (Lonza), 10 μg/ml streptomycin (Lonza) and 50 μg/ml gentamycin (Sigma). Overnight cultured hepatocytes were supplemented with 5% (v/v) fetal bovine serum (Gemini Bio-Products), 140 nM insulin (Thermo Fisher Scientific) for 3 hours and cultured overnight in 14nM insulin. Cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
4
Spheroid Invasion in Collagen Matrix
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MDA-MB-157 and BT-549 cells were stained with 2 μM Calcein AM before harvesting the cells for spheroid formation. An ATPS technology was used to form spheroids [21 (link)]. Spheroids were suspended in an ice-cold 4 mg/ml solution of type I rat tail collagen (Corning) and then incubated at 37 °C for 30 min to allow collagen gelation. Invasion of the spheroids in collagen matrix was captured using fluorescent confocal microscopy (Nikon A1 confocal system) at a 10X magnification. FITC filter was used to capture images with a z-spacing of 20 μm. NIS Elements software was used for image acquisition. Z-projected images were reconstructed by collapsing the stacks using ImageJ (NIH) and total area of pixels was found using color thresholding of the resulting images.
5
3D Membrane-free Microfluidic BMPS Construction
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The OrganoPlate (MIMETAS, Netherlands) was used to construct the 3D membrane-free microfluidic BMPS and the tissue construct method was described previously [4 (link)]. Briefly, collagen solution (Corning, Type I Rat Tail) was diluted in DMEM and the pH was neutralized to 7.0–7.4. Collagen preparation was performed on ice. N2a, C8-D1A, and BV-2 cells were re-suspended in the ECM at the following concentrations: N2a – 3.12 x 106 cells/mL, C8-D1A – 3.12 x 106 cells/mL, and BV-2–1.56 x 106 cells/mL. The cell-ECM mixture was added to the gel lane and incubated (37°C, 5% CO2) for 1 h for gel polymerization. After polymerization, bEnd.3 cells were dispensed into the medium lane at a concentration of 1 × 107 cells/mL in DMEM. The plate was incubated against the side of the incubator at a 75° angle for 4 h to allow bEnd.3 cells to settle against the ECM. Then, 50 μL medium was added to the medium inlet and outlet. The plate was placed on an interval rocker (MIMETAS, The Netherlands) for medium perfusion inside the incubator (37°C, 5% CO2). The rocker (switching between +7° and −7° inclination every 8 min) created a bi-directional flow with a mean flow rate of 2.02 μL/min and a mean shear rate of 0.13 Pa. Medium (50 μL each in the inlet and outlet) was refreshed every other day.
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