293 T cells, which are human embryonic kidney cells engineered with the T-antigen from SV40 to support constant growth, were kindly gifted by Dr. Udaykumar Ranga (JNCASR, Bangalore). TZM-bl cells (Catalog Number 8129), also called JC53-BL, were obtained from the NIH AIDS Reagents Repository. This is a genetically engineered HeLa cell clone that expresses CD4, CXCR4, and CCR5 and contains Tat-responsive reporter genes for firefly luciferase and Escherichia coli β-galactosidase under the regulatory control of an HIV-1 Long Terminal Repeat (37 (link), 38 (link)). Both cell lines were maintained in DMEM (Lonza, Cat# 12-604 F) containing 10% heat-inactivated fetal bovine serum (Lonza) and 50 µg gentamicin/mL (Sigma) in vented T-75 tissue culture flasks (NUNC). Cultures were incubated at 37°C in a humidified 5% CO2–95% air environment. Cell monolayers were split 1:10 at confluence by treatment with 0.25% trypsin, 1 mM EDTA (Sigma Cat# T4049).
T75 tissue culture flask
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United Kingdom, United States
The T75 tissue culture flask is a laboratory equipment used for the cultivation and propagation of cells in vitro. It provides a sterile, temperature-controlled environment for cell growth and maintenance. The flask has a surface area of approximately 75 cm2 and is made of tissue culture-grade polystyrene material.
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Lab products found in correlation
Human ab serum, by Lonza (2 mentions)
Ficoll paque plus, by Cytiva (2 mentions)
Streptomycin, by Lonza (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Rhil 2, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Genios microplate reader, by Tecan (1 mentions)
Iscove modified dulbecco media (imdm), by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Imdm medium, by Lonza (1 mentions)
Ficoll, by Merck Group (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Foetal bovine serum fbs, by Biosera (1 mentions)
Cell death elisa kit, by Roche (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
13 protocols using t75 tissue culture flask
1
Cell Culture of 293T and TZM-bl Lines
2
Evaluating Cytotoxicity of Brown Algae in HT1080 Cells
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Human fibrosarcoma HT1080 cells were grown as monolayers in T-75 tissue culture flasks (Nunc, Roskilde, Denmark) at 5% CO2 and 37°C humidified atmosphere using Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 100 μg/mL penicillin-streptomycin (Gibco-BRL). The medium was changed twice or three times each week.
In order to determine non-toxic concentrations of brown algae samples, the cytotoxic effects were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay in HT1080 cells. Briefly, the cells were cultured in 96-well plates at a density of 5×103 cells/well. After 24 h, the cell culture medium was changed with the control medium or the medium containing samples. After incubation of 48 h, cells were washed and 100 μL of MTT solution (1 mg/mL) was added and incubated for 4 h. Next, in order to solubilize the formed formazan crystals, 100 μL of dimethyl sulfoxide was added and formazan amount was determined by measuring the absorbance at 540 nm using a GENios® microplate reader (Tecan Austria GmbH, Grödig, Austria). Viability of cells was quantified as a percentage compared to the control, and dose response curves were developed.
In order to determine non-toxic concentrations of brown algae samples, the cytotoxic effects were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay in HT1080 cells. Briefly, the cells were cultured in 96-well plates at a density of 5×103 cells/well. After 24 h, the cell culture medium was changed with the control medium or the medium containing samples. After incubation of 48 h, cells were washed and 100 μL of MTT solution (1 mg/mL) was added and incubated for 4 h. Next, in order to solubilize the formed formazan crystals, 100 μL of dimethyl sulfoxide was added and formazan amount was determined by measuring the absorbance at 540 nm using a GENios® microplate reader (Tecan Austria GmbH, Grödig, Austria). Viability of cells was quantified as a percentage compared to the control, and dose response curves were developed.
3
Monocyte Isolation and Differentiation
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All patients donating blood gave informed consent to the Sheffield blood Transfusion Service and all procedures have been approved by the University of Sheffield Ethics Committee. Mononuclear cells were isolated from platelet-depleted buffy coats (Blood Transfusion Service, Sheffield, UK) using Ficoll-Paque Plus (Amersham Pharmacia, St Albans, UK).
In brief, 50 million monocytes were plated into T75 tissue culture flasks (NUNC, UK) and after 2 h non-adherent cells were removed. The remaining adherent cells were cultured over 7 days in IMDM (Lonza, UK) supplemented with 2 mmol l−1L -glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2% human Ab serum (Lonza).
In brief, 50 million monocytes were plated into T75 tissue culture flasks (NUNC, UK) and after 2 h non-adherent cells were removed. The remaining adherent cells were cultured over 7 days in IMDM (Lonza, UK) supplemented with 2 mmol l−1
4
Monocyte Isolation and Differentiation
5
Isolation and Culture of PBMC and Brain Tumor Cells
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Human peripheral blood mononuclear cells (PBMC) were isolated buffy coats obtained from the Sheffield Blood Transfusion Service as previously published 35 (link). Briefly, the PBMC layer was collected following centrifugation over Ficoll (Sigma Aldrich, UK) and cultured overnight in IMDM and 2% human AB serum (Sigma Aldrich, UK). The murine CT-2A - luciferase labelled cells were a gift from Dr Mihaela Lorger (University of Leeds) and the human brain tumour cell lines (U138 and T98G) were purchased from the ATCC. These are established brain tumour cell lines that closely resemble human disease. The murine CT-2A luciferase-labelled cells were kept in a temperature-controlled incubator in T75 tissue culture flasks (NUNC, UK) and cultured with DMEM (Lonza, UK), supplemented with 2 mmol1-1 L- glutamine, 100 U ml-1 penicillin and Foetal bovine serum (FBS, Biosera). Cell lines were routinely mycoplasma tested and genotyped by PCR.
6
Quantifying Apoptosis in Human SCLC Cells
7
Expansion of Bone Marrow Stem Cells
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BMSCs were acquired from PROMOCELL (Heidelberg, Germany). Cells were thawed and expanded according to the protocol previously described by Silva et al. [44 (link)]. Briefly, BMSCs were cultured in α-MEM (GIBCO, Grand Island, NY, USA) supplemented with sodium bicarbonate (NaHCO3, MERCK, USA), 10% of fetal bovine serum (FBS, BIOCHROM AG, UK), and 1% of penicillin-streptomycin antibiotic (GIBCO). Confluent cells were trypsinised, plated in new T75 tissue culture flasks (NUNC, Denmark), at a density of 4.000 cells/cm2, and incubated at 37°C in a 5% humidified CO2 atmosphere. The culture medium was changed every two to three days. BMSCs were used for experiments during passage 6 (P6).
8
Isolation and Characterization of Human Mesenchymal Stromal Cells
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Human bone marrow was harvested from adult donors undergoing intramedullary nailing at the University of Malaya Medical Center after obtaining written informed consent. The methods were followed in accordance with the approved guidelines. All the experimental protocols were approved by the medical ethics committee (Approval ID No. MECID.NO: 201412-859, UMMC). A total of 2 ml of bone marrow was diluted with 2 ml of phosphate-buffered saline (PBS; pH 7.2) and layered onto 3 ml of Ficoll-Paque Premium (GE Healthcare, Sweden) before subjecting to gradient centrifugation at 1800 rpm for 30 min (Eppendorf 5810R). The mononuclear layer was then collected and washed twice with low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen-Gibco, USA) supplemented with 1% antibiotic/antimycotic (v/v; Invitrogen-Gibco). The isolated mononuclear cells were cultured in a growth medium (low-glucose DMEM supplemented with 10% fetal bovine serum, 1% antibiotic/antimycotic (v/v), and 200 mM GlutaMAX TM-I (Invitrogen-Gibco)) and transferred into T75 tissue culture flasks (Nunc TM, USA). The medium was changed on day 5 to remove non-adherent cells, and subsequent medium replacement was conducted at 3-day intervals. The surface markers were examined using a flow cytometer to confirm the characteristics of mesenchymal stromal cells (Supplementary data ).
9
Transient Transfection of HEK293 Cells
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HEK293 (American Tissue Culture Collection, Manassas, VA, USA) were routinely maintained in DMEM containing 10% FBS, 2 mM L-glutamine, pyridoxine, and 110 mg/ml sodium pyruvate. Cells were split every three to six days in a ratio of 1:5 using 0.25% trypsin/EDTA. Cells were plated on T75 tissue culture flasks (Nunc) 24 h prior to transfection, and transfected with plasmid DNA of rTRPV1 (D. Julius, Department of Physiology, University of California, Berkeley, CA, USA), mTRPA1 (A. Patapoutian, The Scripps Research Institute, La Jolla, CA, USA), rTRPM8 (K. Zimmermann, Department of Anesthesiology, Friedrich-Alexander-University, Erlangen-Nuremberg, Erlangen, Germany), hV1A, or hOTR (OriGene Technologies, Rockville, MD, USA) using Lipofectamine 2000 according to the manufacturer's instructions. Twenty-four hours after transfection, cells were plated on 384-well plates and used for Ca2+ experiments 24 h after plating.
10
TZM-bl Cell Line for HIV Infectivity
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The TZM-bl cell line from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc, (Takeuchi et al, 2008; (link)Wei et al, 2002; Derdeyn et al, 2000; Platt et al, 1998) was used in determining toxicity and proliferation. This is an adherent HeLa cell line engineered to stably express CD4, CXCR4 and CCR5 and containing reporter genes controlled by the HIV-1 promoter enabling HIV-1 infectivity determination.
These cells are a model cell line for testing HIV infectivity and was used because of our previous work with HIV and also because we aimed in a separate study to determine the effect of the compounds on HIV infectivity.
The cells were sub-cultured in T-75 tissue culture flasks (Nunc UT, USA). Sub-culturing was done every two or three days when confluency (surface area of the culture flask occupied by cells) was about 90%. Cell culture conditions for the bioassays (MTT and impedance), which included cell number, media volume and incubation time, were established through titration experiments on the RT-CES™ analyser. The pre-determined conditions were maintained for both bioassays since a similarity in the efficiency for viable cell count has been reported for the two (Xing et al, 2006) (link).
These cells are a model cell line for testing HIV infectivity and was used because of our previous work with HIV and also because we aimed in a separate study to determine the effect of the compounds on HIV infectivity.
The cells were sub-cultured in T-75 tissue culture flasks (Nunc UT, USA). Sub-culturing was done every two or three days when confluency (surface area of the culture flask occupied by cells) was about 90%. Cell culture conditions for the bioassays (MTT and impedance), which included cell number, media volume and incubation time, were established through titration experiments on the RT-CES™ analyser. The pre-determined conditions were maintained for both bioassays since a similarity in the efficiency for viable cell count has been reported for the two (Xing et al, 2006) (link).
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