Differentiation of C2BBe1 was performed as previously described by Huang et al. [27 (link)]. Briefly, the C2BBe1 cells were seeded on trans-well inserts (0.4 µm pore size, 12-mm membrane diameter; Corning, Kennebunk, ME, USA) at a density of 5 × 105 cells/cm2 and cultured in 1:1 ratio of MEM/EBSS containing 10% FBS: enterocyte differentiation medium (Corning, Kennebunk, ME, USA). After 24 h, the medium was changed to enterocyte differentiation medium supplemented with 1% ITS-A (Invitrogen, Grand Island, NY, USA) and maintained for at least 5 days.
Its a
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ITS-A is a laboratory instrument designed for the analysis of inorganic and organic compounds. It features a compact and durable design to provide reliable and consistent results. The core function of the ITS-A is to perform high-performance analytical measurements.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Gentamycin, by Thermo Fisher Scientific (5 mentions)
Collagen 4 coated dishes, by Merck Group (4 mentions)
Interferon γ, by R&D Systems (4 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dmem f12 1 1 mixture, by Thermo Fisher Scientific (2 mentions)
Transwell insert, by Corning (1 mentions)
Plc prf 5, by Thermo Fisher Scientific (1 mentions)
Plc prf 5, by American Type Culture Collection (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Sartorius (1 mentions)
Cmrl 1066, by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (1 mentions)
27 protocols using its a
1
Differentiation of C2BBe1 Enterocyte Model
2
Derivation of Transformed Mouse Colon Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Young adult mouse colon (YAMC) cells and derivation of transformed
cells with multiple oncogenic lesions were derived and are used as described
elsewhere (McMurray et al., 2008 (link);
Whitehead et al., 1993 (link)). Briefly,
polyclonal cell populations harboring mutant forms of p53
(p53175H) and Ha-Ras (RasV12) (abbreviated as
mp53/Ras) were derived by retroviral infection of low-passage polyclonal
YAMC cells. YAMC cells were cultured on Collagen IV-coated dishes (1
μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640
medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone),
1 × ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and
5 U/ml interferon γ (R&D Systems). All experiments testing the
effects of genetic perturbations were carried out at the non-permissive
temperature for large T function (39°C) and in the absence of
interferon γ.
cells with multiple oncogenic lesions were derived and are used as described
elsewhere (McMurray et al., 2008 (link);
Whitehead et al., 1993 (link)). Briefly,
polyclonal cell populations harboring mutant forms of p53
(p53175H) and Ha-Ras (RasV12) (abbreviated as
mp53/Ras) were derived by retroviral infection of low-passage polyclonal
YAMC cells. YAMC cells were cultured on Collagen IV-coated dishes (1
μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640
medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone),
1 × ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and
5 U/ml interferon γ (R&D Systems). All experiments testing the
effects of genetic perturbations were carried out at the non-permissive
temperature for large T function (39°C) and in the absence of
interferon γ.
3
Immortalized Mouse Colon Cell Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Low-passage polyclonal young adult mouse colon (YAMC) cells were
originally derived from males and females of the Immorto-mouse
(H-2Kb /tsA58 transgenic mouse) and gifted to the Land
laboratory by a gift from R. Whitehead and A.W. Burgess (D’Abaco et al., 1996 (link); Whitehead et al., 1993 (link)). YAMC cells express
temperature sensitive simian virus 40 large T (tsA58) under the control of
an interferon γ inducible promoter were maintained at the permissive
temperature (33°C) for large T in the presence of interferon γ
to support conditional immortalization in vitro. This
permits expansion of the cells in tissue culture. In contrast, exposure of
YAMC cells to the non-permissive temperature for large T (39°C) in
the absence of interferon g leads to growth arrest followed by cell death,
indicating the absence of spontaneous immortalizing mutations in the cell
population. The cells were cultured on Collagen IV-coated dishes
(1μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640
medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone),
1 × ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and
5 U/ml interferon γ (R&D Systems). All experiments testing the
effects of RasV12 and p53175H were carried out at the
non-permissive temperature for large T function (39°C) and in the
absence of interferon γ.
originally derived from males and females of the Immorto-mouse
(H-2Kb /tsA58 transgenic mouse) and gifted to the Land
laboratory by a gift from R. Whitehead and A.W. Burgess (D’Abaco et al., 1996 (link); Whitehead et al., 1993 (link)). YAMC cells express
temperature sensitive simian virus 40 large T (tsA58) under the control of
an interferon γ inducible promoter were maintained at the permissive
temperature (33°C) for large T in the presence of interferon γ
to support conditional immortalization in vitro. This
permits expansion of the cells in tissue culture. In contrast, exposure of
YAMC cells to the non-permissive temperature for large T (39°C) in
the absence of interferon g leads to growth arrest followed by cell death,
indicating the absence of spontaneous immortalizing mutations in the cell
population. The cells were cultured on Collagen IV-coated dishes
(1μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640
medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone),
1 × ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and
5 U/ml interferon γ (R&D Systems). All experiments testing the
effects of RasV12 and p53175H were carried out at the
non-permissive temperature for large T function (39°C) and in the
absence of interferon γ.
4
Cell line authentication and culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PLC/PRF/5 (CRL-8024) was obtained from American Type Culture Collection (ATCC). CLC7 was a kind gift from Dr. Lijian Hui of Shanghai Institutes for Biological Sciences. HCC cell lines used in this study were authenticated by short tandem repeat (STR) DNA Profiling and no cellular cross-contamination (Figure S10 ) or mycoplasma contamination was detected. PLC/PRF/5 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM-HG, Invitrogen Gibco, Massachusetts, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 1 × (50 unit/ml) penicillin-streptomycin cocktail (P/S) (Gibco, USA). CLC7 cells were cultured in the RPMI1640 medium (Invitrogen Gibco, Massachusetts, USA) supplemented with human recombinant EGF (Thermo Fisher PeproTech, Massachusetts, USA) and ITS-A (Invitrogen Gibco, Massachusetts, USA). Cell lines were cultured at 37°C and 5% CO2 incubator.
5
Isolation and Culture of Bone-Marrow MSCs
6
Immortalized Mouse Colon Cell Line
7
Chondrogenic Differentiation of Expanded Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chondrogenic differentiation was induced by culturing the expanded chondrocytes in pellets using defined serum-free medium as previously described [27 ]. Briefly, cells were re-suspended in chondrogenic medium (BM, 1.25 mg/mL human serum albumin, ITS-A (Invitrogen), 10 ng/mL TGF-ß1 (R&D Systems), and 10-7 M dexamethasone and 0.1 mM ascorbic acid 2-phosphate (Sigma-Aldrich). Aliquots of 2.5 × 105 cells/250 μL were centrifuged at 250 g for 5 minutes in 1.5 mL screw-cap Eppendorf tubes. Pellets were cultured for 2 weeks in a humidified incubator (37 °C, 5 % CO2, 19 % oxygen) with a change of medium twice a week.
8
Chondrocyte Inflammatory Response Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To mimic the OA environment in in vitro cell culture conditions, TC28a2 cells were seeded in the center of the individual wells of 24-well plates at 1×105 cells per well in a 10-µL volume. The cells were then allowed to adhere for 2 h in a cell culture incubator. Next, DMEM-HG supplemented with 1% insulin transferrin selenium-A (ITS-A; Invitrogen, Carlsbad, CA, USA) was added gently. IL-1β (R&D Systems, Minneapolis, MN, USA) was chosen to induce and maintain inflammatory conditions, and the concentration of IL-1β was 10 ng/mL, based on our previous results.15 (link) The cells were maintained for 5 d in the absence or presence of IL-1β and stained with 1% safranin O (Sigma) solution to visualize proteoglycan content produced by the chondrocytes. Crystal violet (CV; Merck, Darmstadt, Germany) staining was performed to compare the density of the cells stained by safranin O. The CV staining method has been described previously.16 (link) For quantitative analysis, absorbance was detected at 490 nm after destaining with 50% acetic acid solution for 20 min. Each safranin O value was normalized to absorbance from CV staining.
9
Derivation of Transformed Mouse Colon Cells
10
Dual Cell Culture Protocol for AML12 and SH-SY5Y
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AML12 cells are a hepatocyte cell line established from male CD1 mice and were obtained from ATCC (Cat# CRL‐2254) and maintained in DMEM/F12 medium (Cat #11320033, Thermo) supplemented with 1x ITS‐A (Invitrogen Cat 51300044), 40 ng/ml dexamethasone and 10% fetal bovine serum (Cat #16140, Thermo). At 24 h prior to experiments, AML cells were switched to a serum‐free formulation. SH‐SY5Y neuroblastoma cells are derived from human female metastatic bone tissue (Biedler et al., 1973 (link)) and were a gift from Dr. Meng‐Yang Zhu (ETSU). SH‐SY5Y cells were differentiated into cholinergic‐like cells by addition of retinoic acid (10 μM) to DMEM supplemented with 1% FBS and 2 mM L‐glutamine for 7 days. These cells were then split and plated in Neurobasal medium supplemented with B‐27 and retinoic acid as described (Shipley et al., 2016 ). AML cells were plated directly onto differentiated SHSY‐5Y cells for 24 h before RNA isolation and gene expression analysis by RT‐qPCR. Knockdown by siRNA was performed as previously described (Keasey et al., 2018 (link)) targeting M1 (Cat #L‐058643‐00‐0005, Horizon Discovery) and M3 (Cat # L‐042172‐00‐0005, Horizon Discovery) receptors or non‐targeting controls (D‐001810‐10‐05).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!