CHO-K1 cells (Sigma-Aldrich) were grown in Petri dishes containing F12 medium (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (Invitrogen) and penicillin-streptomycin-glutamine (100 U/ml, 100 µg/ml, 292 µg/ml) and maintained in a humidified 5% CO2/95% air atmosphere. Cells were electroporated (Nucleofector II; Amaxa) with the plasmids mentioned above (4 µg DNA per reaction) using the Nucleofector kit V (Lonza) according to the manufacturer’s instructions and seeded onto 5-mm-diameter coverslips (Warner Instruments). It is important to note that at least 4 µg DNA was required per electroporation to measure a robust Ae4 activity in CHO-K1 cells 18–20 h after electroporation.
Cho k1 cells
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
CHO-K1 cells are a cell line derived from Chinese hamster ovary (CHO) cells. They are widely used as a model system for studying various cellular and molecular processes. CHO-K1 cells are known for their ability to grow rapidly and efficiently in culture, making them a popular choice for a wide range of applications, such as protein production, drug screening, and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Nucleofector 2, by Lonza (2 mentions)
Nucleofector kit 5, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
5 mm diameter coverslips, by Warner Instruments (1 mentions)
Hitrap mabselect sure column, by GE Healthcare (1 mentions)
Formic acid, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Hplc grade water, by Avantor (1 mentions)
C18 trap column, by Harvard Apparatus (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Quikchange 2 xl, by Agilent Technologies (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Polyfect, by Qiagen (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
9 protocols using cho k1 cells
1
Optimized Ae4 Activity Evaluation
2
Affinity Purification and Protein Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HiTrap Mabselect SuRe column was purchased from GE Healthcare (Chicago, IL, USA) and C18 trap column was purchased from Harvard Apparatus (Holliston, MA, USA). Trypsin for protein digestion was purchased from Promega (Madison, WI, USA). Phosphate buffered saline (PBS), 1,4-dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), and formic acid (FA) were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC-grade water and acetonitrile (ACN) were purchased from J.T. Baker (Phillipsburg, NJ, USA). CHO-k1 cells were purchased from Sigma Aldrich.
3
Characterization of KCNQ1/KCNE1 G229D Mutation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We characterized the effects of the G229D mutation on KCNQ1/KCNE1 (IKs) channel function by whole-cell patch-clamp in a heterologous expression system [Chinese Hamster Ovary-K1 (CHO-K1) cells]. KCNQ1 (GenBank® accession number AF000571 ) and KCNE1 are as described in Harmer et al. (2014) (link). pEGFP-N1 was from Clontech. The patient-identified G229D mutation (c.686G > A) was introduced into KCNQ1 using site-directed mutagenesis [Quikchange II XL (Agilent Technologies)].
CHO-K1 cells (Sigma-Aldrich, 85051005) were cultured as described in Harmer et al. (2014) (link). To analyze the effects of G229D, cells were transfected with 250 ng of wild-type (WT) KCNQ1 or LQT1 mutant cDNA and 500 ng of KCNE1 (+50 ng pEGFP-N1) (IKs-WT or IKs-G229D, respectively). To mimic the heterozygous patient phenotype (IKs-HET), cells were transfected with 125 ng of wild-type channel + 125 ng of mutant channel and 500 ng KCNE1 (+50 ng pEGFP-N1). Transfections were performed as described in Harmer et al. (2014) (link). After transfection, cells were split at low density onto 10 mm glass coverslips and transfected cells (identified by fluorescence) were patched 48 h later.
CHO-K1 cells (Sigma-Aldrich, 85051005) were cultured as described in Harmer et al. (2014) (link). To analyze the effects of G229D, cells were transfected with 250 ng of wild-type (WT) KCNQ1 or LQT1 mutant cDNA and 500 ng of KCNE1 (+50 ng pEGFP-N1) (IKs-WT or IKs-G229D, respectively). To mimic the heterozygous patient phenotype (IKs-HET), cells were transfected with 125 ng of wild-type channel + 125 ng of mutant channel and 500 ng KCNE1 (+50 ng pEGFP-N1). Transfections were performed as described in Harmer et al. (2014) (link). After transfection, cells were split at low density onto 10 mm glass coverslips and transfected cells (identified by fluorescence) were patched 48 h later.
4
Monitoring Intracellular pH in Transfected CHO-K1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO‐K1 cells (Sigma‐Aldrich) were cultured and maintained as previously described (Park et al. 2001 ). Plasmids encoding mouse Slc4a11 (GenBank accession no. NM_ 001081162), human CD8A (GenBank accession no. NM_001768) and the empty pCMV6 entry vector as a transfection control were obtained from OriGene (OriGene Technologies, Rockville, MD). Cells were grown in 10 cm diameter dishes and electroporated (Nucleofector II; Amaxa, Gaithersburg, MD) when 80–90% confluent with targeted plasmids (6 µg each per reaction) using a Nucleofector kit V (Lonza, Alpharetta, GA) according to the manufacturer’s instructions and then seeded onto 5‐mm‐diameter coverslips (Warner Instrument, Hamden, CT). Intracellular pH (pHi) was monitored 18–20 h after electroporation.
5
Plasmid Transfection in CHO-K1 and Ate1 KO Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pEGFP-N2-Ate1-1, pEGFP-N2-Ate1-2, pEGFP-N2-Ate1-3 and pEGFP-N2-Ate1-4 plasmids were kindly provided by Dr. Anna Kashina (Department of Medical Sciences, University of Pennsylvania, Philadelphia, USA). CHO-K1 cells (A.T.C.C., Manassas, VA, USA) were transfected using polyethylenimine (Sigma-Aldrich, St Louis, MO, USA) in serum-free medium with 1 μg of the indicated plasmid per 35 mm-diameter dishes for 3 h at 37 °C. Ate1 KO cells were transfected using Lipofectamine 2000 (Thermo Scientific, Waltham, MA, USA) and Opti-MEM (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
6
CHO K1 Cell Culture and Cryopreservation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO K1 cells were purchased from Sigma-Aldrich (St. Louis, MO) and cultured in 90% F10 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were frozen in freezing medium composed of 80% F10 medium, 10% FBS and 10% dimethylsulfoxide.
7
Transfection of CHOk1 Cells with SCN1A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHOk1 cells (Sigma-Alrich) were grown in Ham’s F-12 nutrient mixture supplemented with 10% FBS (Gibco) at 37°C. Using Polyfect (Qiagen), cells were transfected with 1 μg of SCN1A (wildtype or mutant), 1 μg of eGFP, and 0.5 μg of the β1 subunit. Cells were plated on sterile glass coverslips approximately 24 h after transfection, and experiments were performed 24–48 h later.
8
Measuring KCNC3 Protein Stability in CHO-K1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-K1 cells (Sigma, 85051005) were grown in Ham’s F-12 medium (ThermoFisher, 31765–092) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/mL; ThermoFisher). At 80 to 90% confluency, the cells were cotransfected with 6 µg of pcDNA3 plasmids containing WT Kcnc3, Kcnc3G434V, and 1.2 µg of eGFP plasmids per 10-cm2 dish with Lipofectamine LTX (ThermoFisher, 15338030) following manufacturer’s instructions. Details on the vectors are provided in SI Appendix . The transfection was stopped 6 h later by medium change. At 24 h after transfection, cells were replated in 35-mm wells in triplicate. At 24 h after replating, cells were treated with 20 µg/mL cycloheximide (Sigma, C4859) for 0, 1, 2, 3, 4, and 5 h. At the indicated time, cells were lysed with RIPA buffer, and total protein was subjected to Western blot. The blots were scanned, and signals were measured with ImageStudioLite (LI-COR). The KCNC3 signal of each sample was adjusted with the actin signal, and the relative intensity compared with time 0 was calculated in all replicates to obtain the decay curve. The protein half-life was measured by the one phase exponential decay method in Prism 7 (GraphPad).
9
Glutamine Synthetase-Mediated Adipogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glutamine synthetase (GS), methionine sulfoximine (MSX), Chinese hamster ovary K1 (CHO-K1) cells, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), insulin, and pentobarbital sodium were supplied by Sigma-Aldrich (St. Louis, MO, United States). Biotin-conjugated monoclonal antibodies for ELISA were purchased, including osteoprotegerin (OPG) ab255723, ADPN ab108784 (Abcam, Cambridge, United Kingdom). Antibodies for Western blot were GAPDH ab8245, AdipoR1 ab70362, BMP-2 ab14933 (Abcam, Cambridge, United Kingdom), and ALP sc-271431 and Osteocalcin (OCN) sc-376726 (Santa Cruz, CA, United States). Antibodies used for immunofluorescent staining were BMP-2 ab6285 (Abcam, Cambridge, United Kingdom); ALP sc-271431, OCN sc-390877 (Santa Cruz, CA, United States); and AdipoR1 BM4566 (Boster Bio, CA, United States).
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!