HCET cells (HCE-2 [50.B1], ATCC, Gaithersburg, MA, USA) were cultured in Keratinocyte-serum free medium (Gibco, Grand Island, NY, USA) with 5 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract, 0.005 mg/mL insulin, and 500 ng/mL hydrocortisone as reported before [73 (link)]. HCEC (primary corneal epithelial cells, ATCC) were cultured in Keratinocyte-serum free medium (Gibco) with 5 ng/mL human recombinant EGF and 0.05 mg/mL bovine pituitary extract as per the manufacturer’s protocol. To evaluate the effects of SKQ1, a subset of cells was incubated with 50 nM SKQ1 as previously described [59 (link)], 1 h prior to PM10 exposure. Phase contrast microscopy was used to photograph cell preparations using a Leica EL 6000 microscope (Deerfield, IL, USA).
Keratinocyte serum free medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Germany, Canada, Italy, Switzerland
Keratinocyte serum-free medium is a specialized cell culture medium designed to support the growth and maintenance of keratinocyte cells in an in vitro environment. It provides the necessary nutrients and growth factors required for the proliferation and differentiation of keratinocytes without the need for serum supplementation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (129 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (71 mentions)
Rpmi 1640, by Thermo Fisher Scientific (58 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (45 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (42 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (39 mentions)
Bovine pituitary extract, by BD (33 mentions)
Penicillin, by Thermo Fisher Scientific (31 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (30 mentions)
Streptomycin, by Thermo Fisher Scientific (29 mentions)
Rwpe 1, by Thermo Fisher Scientific (26 mentions)
Rwpe 1, by American Type Culture Collection (26 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (24 mentions)
Du145, by American Type Culture Collection (22 mentions)
Lncap, by American Type Culture Collection (19 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (14 mentions)
Lncap, by Thermo Fisher Scientific (13 mentions)
Dmem medium, by Thermo Fisher Scientific (12 mentions)
Du145, by Thermo Fisher Scientific (12 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (10 mentions)
447 protocols using keratinocyte serum free medium
1
Assessing SKQ1 Effects on Corneal Cells
2
Breast Epithelial Cell Culture and C14orf166 Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Normal human breast epithelial cells (NBEC) were cultured in Keratinocyte serum-free medium (Life Technologies) supplemented with epithelial growth factor, bovine pituitary extract. Breast cancer cell lines including MCF-7, ZR-75-1, T47D, BT549, SKBR3, BT-474, MDA-MB231 and MDA-MB-361 were purchased from American Type Culture Collection and were cultured under conditions specified by the manufacturer.
The CDS sequence of C14orf166 was PCR amplified from cDNA of NBEC and cloned into the pMSCV-puro retroviral vector (indicated as C14orf166), the empty vector was used as negative control (indicated as Vector). Lipofectamine 2000 was used to transfected vectors into the indicated cells according to the manufacturer’s instruction. The same cells transfected empty vector were used as the negative control.
The C14orf166 siRNA (indicated as C14orf166/RNAi) and its cognate control siRNA (indicated as Scramble) were obtained from Guangzhou RiboBio Co (Guangdong, China). 20 nM siRNA were transfected into the indicated cells in six plates using Lipofectamine RNAiMax Reagent (Life Technologies) according to the manufacturer’s instruction.
The CDS sequence of C14orf166 was PCR amplified from cDNA of NBEC and cloned into the pMSCV-puro retroviral vector (indicated as C14orf166), the empty vector was used as negative control (indicated as Vector). Lipofectamine 2000 was used to transfected vectors into the indicated cells according to the manufacturer’s instruction. The same cells transfected empty vector were used as the negative control.
The C14orf166 siRNA (indicated as C14orf166/RNAi) and its cognate control siRNA (indicated as Scramble) were obtained from Guangzhou RiboBio Co (Guangdong, China). 20 nM siRNA were transfected into the indicated cells in six plates using Lipofectamine RNAiMax Reagent (Life Technologies) according to the manufacturer’s instruction.
3
Cell Culture of Lung Cancer Lines
4
Establishing PCa Cell Lines for Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human PCa cell line LNCaP, DU145, and PC3 were purchased from ATCC (Rockville, MD, USA). Cells were maintained in RPMI‐1640 medium (Wako Pure Chemical Company) containing 100 U/mL penicillin and 100 μg/mL streptomycin with 10% FBS (HyClone, Logan, UT, USA). Immortalized RWPE‐1 prostate epithelial cells were also purchased from the ATCC. RWPE‐1 was maintained in keratinocyte serum‐free medium (Life Technologies, Carlsbad, CA, USA). DU145‐derived GCNT2 knockdown cell lines were established by transfection of four different GCNT2siRNA vectors (Data S1). All cells were analyzed by short tandem repeat analysis (BEX, Tokyo, Japan).
5
Generating Vk2/E6E7 Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate the Vk2/E6E7 (Vk2) culture, 60,000 cells were seeded on 0.4 µm pore-sized transwell polystyrene inserts (BD Falcon, Mississauga, ON, Canada), and keratinocyte serum-free medium (Life Technologies, Carlsbad, CA, USA) supplemented with 0.1 ng/mL human recombinant epidermal growth factor (Life Technologies), 0.05 mg/mL of bovine pituitary extract (Life Technologies), CaCl2, and 100 units/mL penicillin/streptomycin (Sigma Aldrich, Oakville, ON, Canada) were added to the apical and basolateral sides of the culture, as described in [41 (link)]. LLI culture was generated by keeping the media on both the apical (300 µL) and basolateral sides (500 µL) (Figure 1 A). For ALI culture, media from the apical side was removed after 24 h of seeding the cells and media was only present in the basolateral side (1000 µL) (Figure 1 B). Media was replenished every 24 h for 10 days of culture.
6
Prostate Cancer Cell Lines and Luciferase Transduction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human prostate cancer cell lines, PC3, DU145, and 22RV1, and the normal prostate epithelial cell line RWPE1, were obtained from ATCC. PC3 and DU145 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS), while 22Rv1 cells were cultured in RPMI-1640 (Life Technologies) supplemented with 10% FBS. RWPE1 cells were cultured in keratinocyte serum-free medium (Life Technologies) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF. PC3, DU145 and 22Rv1 cells were transduced with lentivirus prepared from the pLenti PGK Neo vector to express firefly luciferase and selected with G418 (0.5 mg/ml).
The PC3, DU145, 22Rv1 and RWPE1 cells were transduced with pSLIK lentiviruses encoding Tet-inducible EGFP or Gαt and selected with hygromycin (200–500 μg/ml) to establish stable cell lines.
The PC3, DU145, 22Rv1 and RWPE1 cells were transduced with pSLIK lentiviruses encoding Tet-inducible EGFP or Gαt and selected with hygromycin (200–500 μg/ml) to establish stable cell lines.
7
Oral Epithelial Cell Lines Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GMSM-K human embryonic oral epithelial cell line (a kind gift from Dr. Daniel Grenier; Gilchrist et al., 2000 (link)) and the human immortalized oral epithelial cell (HIOEC) line (Sdek et al., 2006 (link)) were maintained in keratinocyte serum-free medium (Life Technologies, Carlsbad, CA) supplemented with EGF and bovine pituitary extract (Life Technologies). HEPM human embryonic palatal mesenchyme cells (ATCC CRL-1486) were maintained in DMEM (Hyclone, Pittsburgh, PA) supplemented with 10% fatal bovine serum (Hyclone). All the cell lines used in this study were tested for mycoplasma contamination and authenticated by genetic profiling using polymorphic short tandem repeats.
8
Bulbar Conjunctiva Plating Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A step-by-step protocol on the plating of bulbar conjunctiva was performed as previously described [26 (link)]. Briefly, 2 mm × 2 mm explants were cultured at the liquid air surface at 37 °C and 5% CO2 to initiate outgrowth. Outgrowths were submerged in culture medium consisting of supplemented keratinocyte serum-free medium, of which all the components were derived from Life Technologies (Carlsbad, CA, USA); 50 µg/mL bovine pituitary extract, 5 ng/mL recombinant human epidermal growth factor, 10 µg/mL gentamicin, and 1 µg/mL amphotericin B. Gel-forming mucin production, from mRNA expression to intracellular storage and secretion (vide infra), was evaluated after one and two weeks of culture (Table 1 ).
9
Nasopharyngeal Carcinoma Cell Culture and Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell culture was carried out as described in our previous publication [16 (link)]. Briefly, NPC cell lines CNE-1, CNE-2, C666-1, 6-10B, 5-8F and HONE1 were maintained in RPMI 1640 media with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 ℃ in a humidified incubator with 5% CO2. In addition, NP69, an immortalized nasopharyngeal epithelium cell line, was cultured in the defined keratinocyte serum-free medium (Life Technologies) with specific growth factors.
The miR-296-5p mimic, miR-296-5p inhibitor, TGF-β expression plasmids and corresponding control vectors were transfected into the NPC cells with Lipofectamine 3000 reagent as per the manufacturers’ protocols.
The miR-296-5p mimic, miR-296-5p inhibitor, TGF-β expression plasmids and corresponding control vectors were transfected into the NPC cells with Lipofectamine 3000 reagent as per the manufacturers’ protocols.
10
Keratinocyte Serum-Free Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Keratinocyte serum-free medium (KSFM), all the other medium power, Trizol reagent, Lipofectamine 2000, OPTI-MEM, anti-Myc epitope antibodies and zeocin were from Life Technologies (Grand Island, NY., USA). Endothelial growth medium 2 (EGM2) for growing endothelial cells was from Lonza Inc. (Allendale, NJ, USA). All the chemicals, anti-His tag antibodies and Wortmanin were from Sigma-Aldrich Co. (St. Louis, MO, USA). CellTiter 96® AQueous One Solution (MTS kit) was from Promega Corp (Madison, WI, USA). pLKO_AS2.zeo was from National RNAi Core facility in Academia Sinica, Taiwan. Anti-CD15, anti-CD68, and anti-CD11b antibodies were from Leica Biosciences (Richmond, Illinois, USA). S100A9 antibodies and STAT3 inhibitor VI (iSTAT3) were from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-human CD34 antibody was from DakoCytomation Denmark A/S (Copenhagen, Denmark). Anti-S100A8 antibodies, human recombinant S100A9 (recS100A9) protein and anti-CD31 antibodies were from Abcam (Cambridge, MA, USA). Matrigel was from BD Biosciences (San Jose, CA, USA). Pyrrolidine dithiocarbamate (PDTC) was from Tocris Bioscience (Bristol, UK). AZD1480 was from Selleckchem (Houston, TX, USA).
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!