All experiments were performed using the human coronavirus OC43 (HCoV-OC43) (ATCC VR-1558). The HCoV-OC43 virus appears to be a suitable surrogate for SARS-CoV-2, with comparable physical and genomic size [36 (link)], and has been previously used by our group [14 (link)] and others [37 (link)] for ultraviolet radiation efficacy studies. Recent research on SARS-CoV-2 susceptibility with 222 nm radiation on surfaces [20 (link),21 (link)] as well as other studies investigating coronavirus susceptibility [38 (link)] provide further evidence of similar efficacy between coronaviruses. Details of preparation of this virus and its propagation in host WI-38 normal lung cells (ATCC CCL-75) are available in the manuscript by Buonanno et al. (2020) [14 (link)].
Ccl 75
Manufactured by American Type Culture Collection
Sourced in United States
The CCL-75 is a laboratory equipment item used for cell culture applications. It serves as an incubator to provide a controlled environment for the growth and maintenance of cell lines. The CCL-75 maintains temperature, humidity, and carbon dioxide levels to support optimal cell culture conditions.
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Lab products found in correlation
Wi 38, by American Type Culture Collection (7 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Htb 37, by American Type Culture Collection (2 mentions)
Caco 2, by American Type Culture Collection (2 mentions)
Hcov oc43, by American Type Culture Collection (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Ccl 247, by American Type Culture Collection (1 mentions)
D6429, by Merck Group (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Crl 1572, by American Type Culture Collection (1 mentions)
Axio vert a1, by Zeiss (1 mentions)
Htb 161, by American Type Culture Collection (1 mentions)
17 protocols using ccl 75
1
Efficacy of UV Radiation against HCoV-OC43
2
GIST-T1 cells treatment effects
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GIST-T1 cells, characterized in detail by Taguchi et al (15 (link)), were cultured in DMEM and WI-38 cells (ATCC® CCL-75™) in MEM. Both media were supplemented with 10% fetal calf serum and maintained at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The GIST-T1 cells were maintained in the log-growth phase and treatment groups were treated with 30 µM LY294002 (LY294002 group), 10 µM UO126 (UO126 group) or 30 µM LY294002 + 10 µM UO126 (LY+UO group) at 37°C for 24 h. An equal amount of dimethylsulfoxide (DMSO) was added into the control group and incubated at 37°C for 24 h.
3
Generation and Culture of Mouse Embryonic Fibroblasts
4
Cell Culture Protocol for Various Cell Lines
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Cells were grown at 37°C in a humidified incubator under 5% CO2 in the following media: DMEM (D6429; Sigma-Aldrich)/10% fetal bovine serum (FBS; A&E Scientific) for HeLa (AIDS Ref. 153; National Institutes of Health), HEK293 (LifeTech), and FIB364 cells (HeLa cells stably expressing fibrillarin-GFP); MEM (30-2003; American Type Culture Collection [ATCC]/10% FBS for WI-38 cells (CCL-75; ATCC); and McCoy's 5a modified (Sigma-Aldrich)/10% FBS for HCT116 cells (CCL-247; ATCC). All media were supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Life Technologies). The stable cell lines generated in this work were maintained in DMEM (Sigma-Aldrich) supplemented with FBS (10%), penicillin-streptomycin (Pen-Strep; 1%), hygromycin B (100 μg/ml; Invitrogen), and blasticidin (10 μg/ml; Life Technologies). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich.
5
Cell Culture Protocols for Cancer Research
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OC cell lines PA-1 (PA1; (ATCC® CRL-1572™) and NIH:OVCAR-3 (OVCAR3; ATCC® HTB-161™), and a normal lung fibroblast cell line WI-38 (ATCC® CCL-75™) were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified 5% CO2 chamber (HF90; Heal Force Bio-Meditech Holdings Ltd.). Cells were observed under an inverted phase contrat microscope (Zeiss Axiovert. A1; magnification, ×20).
6
Generation and Culture of Mouse Embryonic Fibroblasts
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MEFs were generated by standard methods from wild type, STING-deficient or cGAS-deficient mice. Detailed laboratory protocols are available upon request. All mice were on the C57Bl/6 background and obtained from both genders. Tmem173-/- (STING-deficient) mice were a gift from J Cambier 49 (link). Mb21d1-/- (cGAS knockout first) mice were described in 50 (link). Ifnar1-/- were a gift from C Reis e Sousa and were originally described in 51 (link). This work was performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines for animal care. This work was approved by a project license granted by the UK Home Office (PPL No. 40/3583) and was also approved by the Institutional Animal Ethics Committee Review Board at the University of Oxford. Cells were cultured under 5% CO2 and 5% O2 at 37°C in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin (100 IU/ml)/streptomycin (100 μg/ml), 4.5 g/l D-glucose and 2 mM L-Glutamine. In some experiments MEFs were incubated at ambient O2 or 40% O2. Cells were repeatedly tested for mycoplasma using specific primers. WI-38 cells were purchased from ATCC CCL-75™ and cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) penicillin (100 IU/ml)/streptomycin (100 μg/ml). No method of cell line authentication was used.
7
Decellularization of Human Lung Fibroblasts
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Human lung fibroblasts (WI-38, CCL-75; ATCC) were cultivated on a tissue culture plate (TCP) at a density of 2 × 104 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (1% P/S) under a normal culture condition (5% CO2, 37℃). The medium was replenished every 2–3 day. On day 10, those confluent cells were rinsed twice with phosphate buffered saline (PBS) and subjected to decellularization via dispensing a solution of 0.25% Triton-X 100 and 20mM NH4OH into the plates, followed by PBS washing and subsequent treatment with 50 U/mL DNase I (18,047 − 019; Invitrogen) and 100 µL /mL RNase A (12,091 − 039; Invitrogen) for 2 h at 37℃. After several washing with PBS, the decellularized extracellular matrix ECM was transferred in a conical tube and kept at -20℃ in deionized water (DW) for further use. The entire process was performed in a sterile condition.
8
Decellularization of Fibroblast-Derived Matrix
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Human lung fibroblasts (WI‐38, CCL‐75; ATCC) were cultivated on tissue culture plate (TCP) at the density of 2×104 cells cm−2 for 9–11 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin under normal culture condition (5% CO2, 37 °C). To obtain decellularized fibroblast‐derived matrix (FDM), those cells were rinsed with phosphate buffered saline (PBS) twice and subsequently added with decellularization solution (0.25% Triton‐X 100 and 20 mm NH4OH). After the removal of the treated solution, 50 U/ml DNase I (18047‐019; Invitrogen) and 2.5 µL mL−1 RNase A (12091‐039; Invitrogen) were added and incubated for 1–2 hr at 37 °C. The decellularized FDM was then rinsed with PBS several times and kept at 4 °C for the future use.
9
Cell Culture Conditions for Comparative Analysis
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Human lung fibroblast cell line WI-38 (ATCC® CCL-75™), human non-small cell lung cancer cell line NCI-H1299 (ATCC® CRL-5803™) and human embryonic kidney cell line HEK293 (ATCC® CRL-1573) were obtained from ATCC. The WI-38 cell line was cultured in MEM medium supplemented with 10% fetal bovine serum (FBS), while the H1299 and HEK293 cell lines were cultured in DMEM medium supplemented with 10% FBS. All the cell lines were incubated at 37 °C in an atmosphere of 5% CO2.
10
Preparation of Cell Conditioned Media
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