HEK293 cells stably expressing the human Kv10.1 (HEK-Kv10.1; kindly donated by Dr. Walter Stühmer from the Max Plank Institute in Germany) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; 12,800–017, Gibco) supplemented with 10% fetal bovine serum (FBS; 26140087, Gibco), 1% penicillin-streptomycin (15140122, Gibco), and zeocin (30 μg/ml; R250-01, Invitrogen). HEK293 cells stably expressing the human Nav1.7 (HEK-Nav1.7) were maintained in DMEM/F12 medium (1:1) (12,500–062, Gibco) supplemented with 10% FBS (26140087, Gibco), 1% penicillin-streptomycin (15140122, Gibco) plus blasticidin (10 μg/ml; R210-01, Gibco) and zeocin (200 μg/ml; R250-01, Invitrogen). To express Nav1.7, cells were induced with DMEM/F12 (1:1) (12,500–062, Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin, 1 μg/ml doxycycline (sc-337,691, Santa Cruz Biotechnology) and 3 mM sodium butyrate (sc-202341B, Santa Cruz Biotechnology) for 24 h prior to experiments. HEK293 cells stably expressing Cav3.3 (HEK-Cav3.3; kindly donated by Dr. Juan Carlos Gomora from the Instituto de Fisiología Celular, UNAM were maintained in DMEM (12,800–017, Gibco) supplemented with 10% FBS (26140087, Gibco), 1% penicillin-streptomycin (15140122, Gibco), and geneticin (1 mg/ml; 11,811–031, Gibco). All cells were cultured at 37 °C in a 5% CO2 incubator.
Penicillin streptomycin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Penicillin/streptomycin is a widely used antibiotic solution that combines two antimicrobial agents, penicillin and streptomycin. It is commonly used in cell culture applications to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Doxycycline, by Santa Cruz Biotechnology (1 mentions)
R25001, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Sirna transfection reagent, by Santa Cruz Biotechnology (1 mentions)
Opti mem, by Santa Cruz Biotechnology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Antibodies against p akt, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Santa Cruz Biotechnology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Compound c, by Merck Group (1 mentions)
High glucose, by Thermo Fisher Scientific (1 mentions)
β glycerophosphate, by Santa Cruz Biotechnology (1 mentions)
Alizarin red s, by Carl Roth (1 mentions)
Fetal bovine serum (fbs), by Santa Cruz Biotechnology (1 mentions)
Penicillin, by Fujifilm (1 mentions)
9 protocols using penicillin streptomycin
1
Stable Expression of Ion Channels in HEK293 Cells
2
Cervical Cancer Tissue Collection
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Tumor specimens were collected from 19 cervical cancer patients undergoing surgical treatments. For controls, normal cervical tissue was collected from 10 healthy age-matched volunteers. The clinical details for the patients are provided in Supplemental Table 1 . All patients had not received neo-adjuvant treatment before surgery. Informed consent was obtained from all individuals before sampling and the study was approved by the ethical committee of the Woman’s Hospital School of Medicine Zhejiang University (IRB-20200338-R). All research was performed in accordance with the relevant guidelines and regulations. Human HPV-positive cervical cancer cell lines (SiHa, HeLa) and HPV-negative cervical cancer cells (C33A) were maintained in our laboratory and cultured in RPMI 1640 containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Santa Cruz) at 37 °C with 5% CO2. A mouse cervical cancer (U14) cell line was purchased from the Institute of Materia Medica (Chinese Academy of Medical Sciences, Beijing, China). Cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum.
3
Autophagy Modulation Pathway Analysis
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3-MA, rapamycin, Chloroquine, compound C and Bafilomycin A1 were purchased from Sigma Aldrich (St. Louis, MO, USA). DMEM, Opti-MEM, penicillin-streptomycin, antibody against glyceraldehyde−3-phosphate dehydrogenase (GAPDH), AMPK siRNA, and siRNA Transfection Reagent were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against LC3, p62 and LAMP2 were bought from Abcam (Cambridge, MA, USA), the antibodies against p-AKT, AKT, AMPK, p-AMPK, mTOR and p-mTOR were from Cell Signaling Technology (Danvers, MA, USA), the recombinant active full-length human Akt1 protein (rAkt1) was purchased from Abcam (Cambridge, MA, USA).
4
Osteogenic Differentiation of MenSCs
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Previous studies have demonstrated the osteogenic differentiation potential of MenSCs (29 (link)), thus we assessed the osteogenic differentiation potential of MenSCs. MenSCs (4×104 cells/well) were cultured in 12-well plates. The induction of osteogenic differentiation was initiated when cells reached sub-confluence by adding the osteogenic medium. The osteogenic medium was the DMEM medium supplemented with high glucose (4.5 g/L) (Gibco, Life Technologies), 10% FBS, 50 µg/mL ascorbic acid, 1% penicillin/streptomycin, 10 mmol/L β-glycerophosphate (Santa Cruz), and 0.1 µmol/L dexamethasone for 21 days. Alizarin Red S (Carl Roth) staining was subsequently performed and osteogenic differentiation was evaluated by microscopic observation. Undifferentiated cells were used as controls and the experiments were performed in triplicates.
5
Culturing Human Glioma and Astrocyte Cells
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The human glioma cell line, U251MG, was purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). U-251MG cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Biowest SAS, Nuaillé, France) and penicillin (100 units/mL) streptomycin (100 µg/mL) solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in non-coated flasks. Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C, and the medium was changed every 2–3 days.
Immortalized human normal astrocytes (HASTR/ci35) were kindly provided by Dr. Tomomi Furihata of Chiba University [36 (link)]. HASTR/ci35 cells were routinely grown at 33 °C with 5% CO2/95% air in Gibco® Astrocyte Medium (A1261301, Life Technologies, Carlsbad, CA, USA) supplemented with 1% N2 supplement, 10% fetal bovine serum (FBS), penicillin–streptomycin, and 4 µg/mL blasticidin S (SantaCruz Biotechnology, Inc., Dallas, TX, USA). For HASTR/ci35 cell differentiation, a culture medium without FBS was used, and the culture temperature was set at 37 °C.
Immortalized human normal astrocytes (HASTR/ci35) were kindly provided by Dr. Tomomi Furihata of Chiba University [36 (link)]. HASTR/ci35 cells were routinely grown at 33 °C with 5% CO2/95% air in Gibco® Astrocyte Medium (A1261301, Life Technologies, Carlsbad, CA, USA) supplemented with 1% N2 supplement, 10% fetal bovine serum (FBS), penicillin–streptomycin, and 4 µg/mL blasticidin S (SantaCruz Biotechnology, Inc., Dallas, TX, USA). For HASTR/ci35 cell differentiation, a culture medium without FBS was used, and the culture temperature was set at 37 °C.
6
Modulating Renal Cell Lines with miR-181b and circCSNK1G3
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Human renal epithelial cell line 4120, renal tubular epithelial cell line HK‐2, embryonic kidney cell line 293FT and Human renal carcinoma cells 786‐O, Caki‐1, A498 and ACHN were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Thermo Fisher Scientific.) with 10% FBS (Gibco) and 1% penicillin/streptomycin (Santa Cruz) at 37°C with 5% CO2.
For cell transfection, ACHN cells were transfected with plasmid containing miR‐181b inhibitor or miR‐181b mimics for miR‐181b knockdown or overexpression; shcircCSNK1G3 or pcDNA3.1‐circCSNK1G3 for circCSNK1G3 knockdown or overexpression using Lipofectamine 2000 reagent (Invitrogen). The shRNA oligo sequence was listed in Table1 . Plasmids used in this research were synthesized by GeneChem (Shanghai, China).
For cell transfection, ACHN cells were transfected with plasmid containing miR‐181b inhibitor or miR‐181b mimics for miR‐181b knockdown or overexpression; shcircCSNK1G3 or pcDNA3.1‐circCSNK1G3 for circCSNK1G3 knockdown or overexpression using Lipofectamine 2000 reagent (Invitrogen). The shRNA oligo sequence was listed in Table
7
Tenogenic Differentiation of hASCs in Hybrid Constructs
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hASCs were encapsulated in the gel layer of TenoForce or BioTenoForce, and a set of experiments were performed to characterize cell viability, proliferation, and tenogenic differentiation. In brief, hASCs (passage 2–5, 4 million/mL) were mixed with GelMA prepolymer containing either 20% (v/v) tECM (final concentration in gel: 0.6 mg/mL) as BioTenoForce group or 20% (v/v) PBS as TenoForce group to formed hybrid constructs by UV irradiation (365 nm, 25 mW/cm2, 90 s). Different groups of scaffolds were cultured in growth medium (DMEM-high glucose (Thermo Fisher), 10% (v/v) FBS, 1% (v/v) Penicillin/Streptomycin, and 50 ng/mL ascorbic acid (Santa Cruz)). On designated time points (i.e., days 0, 7, 10, and 14), Live/Dead (Invitrogen) and dsDNA (Quant-iT PicoGreen dsDNA Reagent, Invitrogen) assays were performed to determine cell viability and dsDNA content, respectively. To evaluate hASC tenogenic differentiation, tenogenesis-associated markers (SCX, MKX, TNC, and COL1A1) were assessed via qPCR and (TNC, COL1, TNMD, and F-actin) via fluorescence staining as previously described [31 (link)]. A detailed description can be found in the supplementary materials.
8
Generating Inducible E2F1 Cell Lines
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Hemagglutinin (HA)–tagged WT, the arginine to lysine 111/113 mutant E2F1 (KK), and the arginine to lysine 109 (R109K) constructs have been described previously (11 (link)). These were subcloned into a pTRE2-hyg expression vector (Clontech) and transfected into parental Tet-On U2OS cells (Clontech; RRID: CVCL_V335) to generate inducible, stable cell lines. These cells were selected in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin/streptomycin, G418 (100 μg/ml; Santa Cruz Biotechnology), and hygromycin B (150 μg/ml; TOKU-E). For all experiments, doxycycline (1 μg/ml) was used to induce protein expression for 24 hours before harvest. E2F1 and TSN CRISPR cells were generated as per the protocol described (28 (link)) and cultured in DMEM containing 10% (v/v) FBS and penicillin/streptomycin. All cell lines were tested for mycoplasma contamination before use.
9
Hedgehog Signaling Pathway Analysis
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Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, antibodies for SHH (Human, pAd), PTCH (Human, pAd), SMO (Human, pAd), Gli1 (Human, pAd), MMP2 (Human, pAd), MMP9 (Human, pAd), TIMP1 (Human, pAd), and TIMP2 (Human, pAd) were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). All antibodies were diluted 1:100. Matrigel was purchased from BD Biosciences (Bedford, MA). Eight-micrometer pore size transwell inserts were purchased from Corning Corporation (Corning, NY). All other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St Louis, MO).
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