PRF and CGF were prepared as stated in previous studies.3 (link),13 (link) In brief, 40 mL of blood was drawn from the forelimb vein and gently pushed into four 10 mL venous blood collection tubes (BD, Franklin Lakes, NJ, USA) without anticoagulant. Two tubes, containing 20 mL blood, were immediately centrifuged at 3000 rpm for 12 min using a HSIANGTA centrifuge (HSIANGTA, New Taipei City, Taiwan). After discarding the red blood cells, the PRF and the remaining serum were collected and immediately stored at −80 °C. Another two tubes containing 18 mL blood were centrifuged using MEDIFUGE centrifuge (MEDIFUGE MF 200 100, Sofia, Italy) at ∼0–2700 rpm for 20 s, 2700 rpm for 2 min, 2400 rpm for 4 min, 2700 rpm for 2 min, 3000 rpm for 3 min, and then stopped from 3000 to 0 rpm in about 36 s to obtain the CGF. Similar to that for PRF, the CGF and the serum were stored at −80 °C until use, except part of PRF and CGF samples were immediately taken for the tensile strength measuring. For those frozen samples, after defreezing and diluting (using phosphate-buffered saline or culture media (Leibovitz L-15, Invitrogen, Grand Island, NY, USA), 1 mL/g weight), the fibrin clots were sonicated (BRANSON, Danbury, CT, USA) with the frequency of 20/min for 3 min. The supernatants were collected after centrifuging (12,000 rpm for 10 min at 4 °C) to determine the growth factor concentrations.
Leibovitz l 15
Manufactured by Thermo Fisher Scientific
Sourced in United States
Leibovitz L-15 is a cell culture medium formulated to support the growth and maintenance of various cell types. It is designed to mimic the in vivo environment and provides essential nutrients, vitamins, and other components required for cell proliferation and survival. The medium is optimized for use in cell culture applications, though its specific intended uses are not included in this factual description.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Medium 199, by Corning (2 mentions)
Zeocin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Sw620, by Thermo Fisher Scientific (2 mentions)
Sw620, by American Type Culture Collection (2 mentions)
Panc 1, by Thermo Fisher Scientific (2 mentions)
Panc 1, by American Type Culture Collection (2 mentions)
Mcdb153, by Merck Group (2 mentions)
Axio observer 7 microscope, by Zeiss (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Mouse gamma interferon, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Sw13 cells, by American Type Culture Collection (1 mentions)
24 protocols using leibovitz l 15
1
Preparation and Analysis of PRF and CGF
2
Isolation and Characterization of Colon Cancer Cells
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P1 cancer cells were established from liver metastasis lesions of colon cancer (10 (link)) and were cultured in RPMI 1640 (Invitrogen, Grand Island, NY) medium containing 10% FBS. The colon cancer cell line SW480 was maintained in Leibovitz L-15 (Invitrogen, Grand Island, NY) medium with 10% FBS; 293T cells were maintained in DMEM (Invitrogen, Grand Island, NY), supplement with NaHCO3 1.5 g/L, medium with 10% FBS. The SW480 and 293T cell line was obtained from the Cell Bank of Chinese Academy of Science, Shanghai. Tumor samples were collected from patients undergoing surgery at the Department of Zhejiang Provincial People's Hospital.
This study was approved by the ethics committees of the Zhejiang Provincial People's Hospital (KY2014-AF-09). Informed consent was obtained from all of the enrolled subjects, and the study was performed in full compliance with all principles of the Helsinki Declaration.
This study was approved by the ethics committees of the Zhejiang Provincial People's Hospital (KY2014-AF-09). Informed consent was obtained from all of the enrolled subjects, and the study was performed in full compliance with all principles of the Helsinki Declaration.
3
Cell Culture Protocols for HEK293, SW13, and Mouse Epicardial Cells
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HEK293 cells (ATCC CRL-1573) were cultured in DMEM/F12+Glutamax supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies); SW13 cells (ATCC CLL-105) were cultured in Leibovitz L-15 (Life Technologies) supplemented with 10% FBS and penicillin-streptomycin; mouse primary epicardial cells were culture in DMEM+Glutamax supplemented with 10% heat-inactivated FCS, 1% streptomycin (Life Technologies), insulin-transferrin-selenium (Biosource) and 10 units ml−1 mouse gamma interferon (PeproTech). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C (HEK293 and SW13 cells) or 33 °C (mouse primary epicardial cells) and were passaged at 80–90% confluence using 0.05% trypsin (Life Technologies). All cell lines used in this study have been routinely tested and found to be negative for mycoplasma contamination.
4
Viral Neutralization Antibody Assay
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Neutralization of viral infectivity test was performed to measure neutralizing antibodies in the serum. A monolayer of MDCK cells was incubated at 37 °C and 5% CO2 overnight in six-well plates at a density of 4.75 × 106 in 3 mL of RP10. The RDE-treated sera prepared as described above were serially diluted two-fold in PBS in a 96-well plate and incubated with 100 PFU of the virus antigens (CA/09 or SG/15) for antigen–antibody reaction. After 60 min, the mixture of virus and serum was added to MDCK cells and incubated at 37 °C and 5% CO2 for 45 min with shaking every 15 min to allow the non-neutralized virus to adsorb to the cells. Warmed L15 overlay medium (consisting of Leibovitz L-15 (Life Technologies Corp., Carlsbad, NY, USA) with glutamine at pH 6.8 supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 0.028% (w/v) NaHCO3, 1 mg/mL TPCK-treated trypsin (Worthington Biochemical Corp., Lakewood, NJ, USA), and 0.9% (w/v) agarose) was added to the six-well plates. The plates were incubated at 37 °C with 5% CO2 for 3 days. Following manual counting of the plaques, the neutralizing antibody titer was determined as the reciprocal of the highest dilution that prevented the growth of plaques to 50% of that obtained in the control wells.
5
Isolation and Culture of NSCs from Mice
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NSCs were isolated from the subventricular zone (SVZ) of mice 2 weeks after the injection of either tamoxifen (tNSC0) or oil (ctrlNSCs) according to [35 (link)]. Glioma-bearing mice displaying termination criteria such as loss of >20% body weight, neurological deficits or poor general condition were euthanized with carbon dioxide; brains were minced and dissociated in Leibovitz-L15 (Life Technologies, Pittsburgh, PA, USA) containing 10 U/mL papain, 5 mM EDTA, and 200 U/mL DNAse. Cells were grown according to [35 (link)].
6
SARS-CoV-2 Neutralizing Antibody Assay
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The neutralization assay was performed to measure neutralizing antibody titers in the serum of vaccinated mice as previously described for influenza viruses42 (link),43 (link). To measure neutralizing antibody titers against SARS-CoV-2, monolayers of Vero TMPRSS2 cells were prepared in 6-well plates by seeding 1.425 × 106 cells/well in 3 mL DMEM and incubated overnight at 37 °C in 5% CO2. Mouse sera were inactivated at 56 °C for 30 min and serially diluted two-fold in 96-well microplates to which 100 PFU of SARS-CoV-2 was added and incubated for 1 h at room temperature. Following washing of Vero TMPRSS2 cells with DMEManti, (containing 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 20 μg/mL of gentamicin) the virus-serum mixture was transferred to 6-well plates containing the Vero TMPRSS2 cells and incubated at 37 °C in 5% CO2, with shaking every 15 min. Finally, warmed L15 overlay medium [consisting of Leibovitz L-15 (Life Technologies Corp., Carlsbad, NY, USA) with glutamine supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.028% (w/v) NaHCO3 and 0.9% (w/v) agarose] was added to the 6-well plates. The plates were incubated for three days at 37 °C in 5% CO2 and the neutralizing antibody titer was determined as the reciprocal of the highest dilution that prevented the growth of plaques to 50% of that obtained in the control wells.
7
Culturing and Titrating Flavivirus Strains
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C6/36 cells were grown in Leibovitz L15 (Life Technologies, The Netherlands) medium, which was supplemented with 10% FBS. Vero E6 cells were cultured with DMEM Hepes (Life Technologies, The Netherlands)-buffered medium supplemented with 10% FBS containing penicillin (100 IU/ml) and streptomycin (100 μg/ml). When Vero E6 cells were infected with mosquito lysates or saliva the growth medium was supplemented with fungizone (2.5 μg/ml) and gentamycin (50 μg/ml). This medium will be referred to as fully supplemented medium. Passage 2 (P2) virus stocks of USUV, Bologna '09 (GenBank accession no. HM569263 ) [26] and WNV Gr'10 lineage 2 (GenBank accession no. HQ537483.1 ) [27] (link), [28] (link) were grown on C6/36 cells and titrated on Vero E6 cells.
8
Chicken Embryo Cell and DF-1-Cre Fibroblast Cultures
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All cells were maintained at 38 °C in a humidified atmosphere of 5% CO2. Chicken embryo cells (CEC) were prepared from 10 to 11-day-old specific-pathogen-free (SPF) embryos obtained from the University of Illinois at Urbana-Champaign (UIUC) Poultry Farm following standard methods42 . Briefly, primary CEC cultures were seeded in growth medium consisting of Medium 199 (Cellgro, Corning, NY, USA) supplemented with 10% tryptose-phosphate broth (TPB), 0.63% NaHCO3 solution, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), and 4% fetal bovine serum (FBS). Confluent CEC cultures were maintained in Medium 199 supplemented with 7.5% TPB, 0.63% NaHCO3, 0.2% FBS, and antibiotics.
The chicken DF-1-Cre fibroblast cell line43 (link) was cultivated in a 1:1 mixture of Leibovitz L-15 and McCoy 5A (LM) media (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), and maintained in 50 µg/ml Zeocin (Invitrogen, Carlsbad, CA).
The chicken DF-1-Cre fibroblast cell line43 (link) was cultivated in a 1:1 mixture of Leibovitz L-15 and McCoy 5A (LM) media (Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), and maintained in 50 µg/ml Zeocin (Invitrogen, Carlsbad, CA).
9
Culturing and Cryopreserving ZFL Cell Line
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ZFL was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). The cell line was cultured in a medium consisted of 50% Leibovitz L-15 (Gibco BRL Co. Ltd. Gaithersburg, MD, USA), 15% Ham F-12 (Gibco) and 35% Dulbecco’s modified eagle medium (DMEM; Gibco), supplemented with 50 ng/mL epidermal growth factor (EGF; Gibco), 0.01 mg/mL insulin (Sigma), 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco) and 5% fetal bovine serum (FBS; Gibco) at 37 °C and saturated with 5% CO2 in a humidified atmosphere. Next, 10% dimethyl sulfoxide (DMSO) and 90% FBS were suspended as cell freezing medium. All the ZFL cells were recovered in a conventional method and observed [23 (link)]. The ZFL cells were seeded on 96-well plates with a cell density of 1 × 104 per well and incubated overnight to achieve a 85% confluence, and then the cells were sub-cultured and frozen.
10
Cell Culture Protocols for Virus Research
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Vero, HeLa, HeLa G3BP1/2 KO (Visser et al., 2019 (link)), U2OS, and U2OS G3BP1/2 KO cells were cultured in Dulbecco's modified Eagle medium (DMEM; Life technologies, Fisher Inv.) containing 10% fetal bovine serum (FBS; Life technologies, Fisher Inv.) and 1% Penicillin/Streptomycin in T25 flasks at 37°C with 5% CO2. Aedes albopictus mosquito C6/36 cells were cultured in Leibovitz (L15) medium (Gibco) containing 1% non-essential amino acids (NEAAs, Thermo Scientific), 2% tryptose phosphate broth (TPB, Gibco), and 10% FBS. Cells were cultured at 28°C in T25 flasks.
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