Human prepuce skin biopsies were cut into fragments of about 3 × 3 mm and placed in a 30 mm dish containing 3–4 ml of PBS with antibiotics and 2 U/ml of Dispase (Sigma-Aldrich, St. Louis, USA) overnight at 4 °C. The epidermal layer was then carefully peeled off using sterile forceps, and immediately washed with PBS/antibiotics before further digestion with 0.05% trypsin for 30 minutes at 37 °C to obtain keratinocyte suspensions. After washing with hepes buffered saline (Lonza, Walkersville, USA) and centrifugation at 176 × g for 5 minutes at RT, the cells were cultured in a keratinocyte growth medium (Lonza, Walkersville, USA), in T25 flasks at 37 °C for 2–3 weeks, changing medium every 2 days.
Hepes buffered saline
Manufactured by Lonza
Sourced in Switzerland
HEPES buffered saline is a laboratory solution that maintains a stable pH environment. It serves as a buffer, helping to maintain the desired pH levels in various cell culture and biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Lonza (2 mentions)
Fibroblast growth factor (fgf), by Lonza (2 mentions)
Insulin, by Lonza (2 mentions)
Ga 1000, by Lonza (2 mentions)
Dispase, by Merck Group (1 mentions)
Keratinocyte growth medium, by Lonza (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Abcam (1 mentions)
Human ventricular cardiac fibroblasts, by Lonza (1 mentions)
Cc 2904, by Lonza (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Pen strep, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Nahco3, by Lonza (1 mentions)
Fastprep 24 5g homogenizer, by MP Biomedicals (1 mentions)
5 protocols using hepes buffered saline
1
Isolation and Culture of Human Keratinocytes
2
Calcium-Dependent Fluo-4 Fluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cells were incubated with 4.5 μM Fluo-4 AM (Thermo Fisher Scientific) in D-PBS (-) for 60 minutes at 37°C. Following the loading, the cells were harvested by centrifugation and resuspended in HEPES-buffered saline (Lonza) containing 10 mM CaCl2. A 3.0 μl droplet containing 1.0×105 Fluo-4-loaded Jurkat cells suspended in HEPES-buffered saline containing 10 mM CaCl2 was added to the silicone oil, and droplet bouncing or short-circuiting was performed. Following the droplet actuation, the droplet was recovered into a microcentrifuge tube containing HEPES-buffered saline without CaCl2. The fluorescence intensity of the cells was measured using flow cytometry.
3
Cardiac Fibroblast Culture and Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ventricular cardiac fibroblasts (Lonza, Basel, Switzerland) were cultured in fibroblast basal medium supplemented with 0.1% insulin, 0.1% fibroblast growth factor, 0.1% GA-1000, and 10% FBS (all Lonza, Basel, Switzerland). Cultures were washed with HEPES buffered saline (Lonza, Basel, Switzerland) when indicated, and split at a confluency level of 70%. Cells were serum starved for 24 h, with subsequent treatment with one of the following: (1) No treatment—control; (2) 20 ng/mL TGFβ (Abcam, Cambridge, UK); or (3) recombinant human tenascin C (1 µg/mL rh TN-C, Fisher Scientific, Waltham, MA, USA) for an additional 24 h. Then, the total RNA was extracted as described previously [19 (link)].
4
Cardiac Fibroblast Culture and Transcription Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ventricular cardiac fibroblasts (HVCFs, cryopreserved ampules of normal human ventricular cardiac fibroblasts containing ≥ 500,000 cells, #CC-2904, Lonza, Basel, Switzerland, https://bioscience.lonza.com/lonza_bs/CH/en/Primary-and-Stem-Cells/p/000000000000197234/NHCF-V-%E2%80%93-Human-Ventricular-Cardiac-Fibroblasts ) were cultured in a fibroblast basal medium supplemented with 0.1% insulin, 0.1% fibroblast growth factor, 0.1% GA-1000, 1% pen-strep, and 10% FBS (all Lonza, Basel, Switzerland) as previously described40 (link),57 (link). Cultures were washed with HEPES buffered saline (Lonza, Basel, Switzerland) when indicated and split at a confluency level of 70%40 (link),57 (link). Cells were treated as follows: (i) without treatment—control; (ii) 20 ng/mL transforming growth factor-beta (TGF-β, R&D systems, Minneapolis, Minnesota, USA); (iii) 10 nmol/L P234; and (iv) 20 ng/mL TGF-β with 10 nmol/L P234 for 24 h. Then total RNA was extracted from HVCFs, and gene expressions of Col1a1, Mmp9, and Acta2 were calculated relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and hypoxanthine–guanine phosphoribosyltransferase (Hgprt) expressions (the specific primer sequences and the description of the RT-qPCR method from HVCFs are detailed in the Supplementary Material ).
5
Mosquito Homogenization and Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mosquito pools were homogenized in 1 ml cold medium (DMEM with 4.5 g/l glucose (Lonza, Verviers, Belgium), NaHCO3 (0.075%, Lonza, Verviers, Belgium), Hepes buffered saline (17 mM, Lonza, Verviers, Belgium), penicillin/streptomycin (1000 U/ml, Lonza, Verviers, Belgium), amphotericin (0.0125 mg/ml, in house made)) with one 1/4″ ceramic sphere (MP Biomedicals, Solon OH, USA) using the FastPrep-24 5G Homogenizer MP Biomedicals (30″ 5 m/s). Mosquito pools were placed at +4 °C for at least 5 min before spinning down the samples for 1 min at 13,000 g.
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!