The normal human colon mucosal epithelial cell line (NCM460) [21 (link)] used in our studies was obtained from INCELL (San Antonio, TX, USA) and maintained in DMEM (with 1000mg/L glucose and without L-glutamine, sodium bicarbonate, folate) medium (D2429, Sigma-Aldrich, MO, USA) supplemented with 15% (v/v) KnockOutTM Serum Replacement (10828–028, Thermo Fisher Scientific, CA, USA), and Penicillin-Streptomycin (100×) (15140–122, Thermo Fisher Scientific, CA, USA). NCM460 was maintained for 8 weeks in medium containing normal (4.0 mg/L) or low (0.4 mg/L) concentrations of folate according to the instructions of the medium.
Knockout serum replacement
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
KnockOutTM Serum Replacement is a serum-free, animal component-free medium supplement designed for the culture of embryonic stem cells and induced pluripotent stem cells. It supports the maintenance of pluripotency and self-renewal of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Non essential amino acids (neaa), by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
β mercaptoethanol, by Merck Group (6 mentions)
Knockout dmem, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (4 mentions)
2 mercaptoethanol, by Merck Group (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Collagenase type 4, by Thermo Fisher Scientific (2 mentions)
Accutase solution, by Merck Group (2 mentions)
Matrigel, by BD (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
28 protocols using knockout serum replacement
1
Culturing NCM460 Cells with Folate
2
Differentiation of iPSCs to Sensory Neurons
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Differentiation of the iPSCs into sensory neurons was adapted from a previous study.10 (link) Briefly, Essential 8TM Flex medium was replaced by knockout serum replacement medium (KSR) consisting of KnockOutTM DMEM (10829018, Thermo Fisher), KnockOutTM Serum Replacement (10828028, Thermo Fisher), GlutaMAXTM, non-essential amino acids (11140050, Thermo Fisher) and β-mercaptoethanol. RevitaCellTM was used to enhance cell survival. SB431542 and LDN-193189 were added to the KSR medium to promote anterior neuro-ectoderm specification. On Day 2, CHIR99021, DAPT and potent and selective vascular endothelial growth factor receptor and fibroblast growth factor receptor inhibitor SU5402 (SU, 3300, Tocris) were supplemented to the KSR medium. The medium was then progressively transitioned into neuronal medium consisting of neurobasal medium, N2 supplement, B-27TM supplement (without vitamin A), GlutaMAXTM and β-mercaptoethanol. Additional compounds used to promote neural crest cells and sensory neuron differentiation and maturation were: ascorbic acid, β-nerve growth factor (450-34, Peprotech), neurotrophin-3 (450-03, Peprotech), BDNF and GDNF. Sensory neurons were replated on Matrigel®-coated plates at a density of 2.5 × 105 cells/ml. Additionally, as in our motor neuron protocol, a single treatment with 1 µM Ara-C was performed on Day 14.
3
Tumor Spheroid Culture and Inhibitor Screening
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Tumor spheroid cells were cultured in growth medium contained with DMEM/F12 (Hyclone, #AC10932967) medium supplemented with 10% KnockOutTM Serum Replacement (Thermo Fisher Scientific, #10828028), 1X non‑essential amino acids (Solaribio, China, #N1250-100), 1 mM sodium pyruvate (Hyclone, #AZH193209), 20 ng/ml human recombinant epidermal growth factor (Thermo Fisher Scientific, #RP-8661), and 10 ng/ml basic fibroblast growth factor (Thermo Fisher Scientific, #RP-8626) for 48 h45 (link). Next, tumor spheroids were cultured in growth medium added with Matrigel (1:4 dilution, n = 3), different inhibitors, and MLT for 48 h. The diameters of tumor spheroids were measured47 . The experiments were replicated three times.
4
Murine Sperm Capacitation and IVF
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Sperm was squeezed out of the cauda epididymis, which was dissected from 2-month-old BDF1 male mice. The retrieved sperm was transferred immediately into a pre-warmed drop of Human Tubal Fluid media (HTF; FUJIFILM Irvine Scientific, San Diego, CA, USA) with 10% KnockOutTM serum replacement (Thermo Fisher Scientific) for sperm capacitation for 1 h. Sperms and oocytes were then inseminated in HTF media for 6 h. Finally, the zygotes were cultured in KSOM medium. The rates of fertilization (number of 2-cell embryos/survived oocytes), cleavage (number of 4-cell embryos/survived oocytes), and blastocyst formation (number of blastocysts/survived oocytes) were evaluated between experimental groups.
5
Mouse Sperm-Oocyte in vitro Fertilization
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Sperm was collected from the cauda epididymis of 12-week-old male mice. Mature MII oocytes in HTF containing 10% KnockOutTM serum replacement (Thermo Fisher Scientific, Fair Lawn, USA) were inseminated with capacitated spermatozoa (1–2 × 106/mL) and incubated for 6 h. Fertilized oocytes were transferred to COOK medium (COOK, Queensland, Australia), covered with mineral oil, and incubated at 37 °C in a humidified 6% CO2 atmosphere. The cleavage rate was recorded on day 2, and the number of embryos that reached the blastocyst stage (hatching and/or hatched blastocyst) was assessed on day 5.
6
Cell Culture and JQ1 Treatment
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MDA-MB-231 breast cancer cells (HTB-26, ATCC) were cultured at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium (Thermo Fisher) supplemented with 10% fetal bovine serum. MM.1S cells were cultured at 37°C and 5% CO2 in RPMI-1640 supplemented with 1% GlutaMAX (Thermo Fisher) and 10% fetal bovine serum. For JQ1 treatment, MDA-MB-231 cells were re-suspended in fresh media containing 500 nM JQ1 (a gift from Cheng-Ming Chiang, UT Southwestern) or 0.05% DMSO as vehicle for a duration of 6 hrs. Mouse ES cells (C57BL/6) were cultured in KnockoutTM DMEM (Thermo Fisher) supplemented with 15% KnockoutTM Serum Replacement (Thermo Fisher), 2 mM L-glutamine (Thermo Fisher), 1× non-essential amino acids (Thermo Fisher), 0.1 mM 2-mercaptoethanol (Thermo Fisher), 1,000 U /ml LIF (Millipore), 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent). Drosophila S2 cells were cultured at room temperature and ambient CO2 in Schneider’s Drosophila Medium (Thermo Fisher) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Thermo Fisher). The origins, authentication and mycoplasma-testing methods of the cell lines used in the current study were listed in the Life Sciences Reporting Summary.
7
Cell Culture and JQ1 Treatment
8
Rat Cardiomyocyte Culture for SECM
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All animal procedures were performed in accordance with the "Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions" stipulated by the Ministry of Education, Culture, Sports, Science and Technology and approved by the animal experiment committee of Cosmo Bio Co., Ltd. Primary rat cardiomyocytes were cultured as previously described. 15 Briefly, cardiomyocytes prepared from neonatal rat ventricles (Cosmo Bio, Japan) were seeded on 35-mm dishes that were coated with collagen at a concentration of 2×10 5 cells/dish. These cells were then incubated in cardiomyocyte culture medium (Cosmo Bio) at 37℃ in a humidified atmosphere containing 5% CO 2 . The medium was changed two days after seeding.
For SECM experiments, we used spontaneously beating cells within 25 days in a culture. Before making a beating measurement, the culture medium was replaced with 2 ml/dish of serum-free medium that contained 1 mM potassium ferrocyanide (K 4 [Fe(CN) 6 ]) that is an electron mediator of SECM measurement and 2% Knockout TM serum replacement (Thermo Fisher Scientific, USA) in DMEM/F-12 medium (Thermo Fisher Scientific, USA).
For SECM experiments, we used spontaneously beating cells within 25 days in a culture. Before making a beating measurement, the culture medium was replaced with 2 ml/dish of serum-free medium that contained 1 mM potassium ferrocyanide (K 4 [Fe(CN) 6 ]) that is an electron mediator of SECM measurement and 2% Knockout TM serum replacement (Thermo Fisher Scientific, USA) in DMEM/F-12 medium (Thermo Fisher Scientific, USA).
9
Maintaining Human Embryonic Stem Cells
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Genome-editing experiments were performed in WIBR#3 hESCs (Lengner et al., 2010 (link)), NIH stem cell registry # 0079. Cell culture was carried out as described previously (Soldner et al., 2009 (link)). Briefly, all hESC lines were maintained on a layer of inactivated mouse embryonic fibroblasts (MEFs) in hESC medium (DMEM/F12 [Lifetech]) supplemented with 15% fetal bovine serum [Lifetech], 5% KnockOutTM Serum Replacement [Lifetech], 1 mM glutamine [Lifetech], 1% non-essential amino acids [Lifetech], 0.1 mM β-mercaptoethanol [Sigma], 1000 U/ml penicillin/streptomycin [Lifetech], and 4 ng/ml FGF2 [Lifetech]. Cultures were passaged every 5–7 days either manually or enzymatically with collagenase type IV [Lifetech] (1.5 mg/ml) and gravitational sedimentation by washing 3 times in wash media (DMEM/F12 [Lifetech] supplemented with 5% fetal bovine serum [Lifetech], and 1000 U/ml penicillin/streptomycin [Lifetech]).
10
mESC Culture and Differentiation
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In all experiments (except viability assay), R1 Oct4 enhanced GFP mouse embryonic stem cells (mESC) (kindly provided by the Zandstra lab) were routinely cultured on gelatin-coated dishes in medium containing 15% serum (Hyclone) and mouse 103 U ml−1 LIF (ESGRO, Millipore). Twelve hours before the experiment, the medium was changed to serum-free knockout medium KnockOutTM Serum Replacement, Life Technologies.
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