All cell culture lines were incubated at 37°C, 5% CO2, 95% humidity in incubators (Thermofisher, FORMA STERI-CYCLE i160) in DMEM medium (Gibco,1199506) with 10% FBS (Gibco,1009914) and 1% penicillin streptomycin (Gibco, 15140122). The identity of the cell lines was not authenticated, and mycoplasma was not determined. Cell were split to 50% density and transfected with Lipofectamine (Invitrogen, 11668–019) 12 hr later. Expression for the transfected constructs was evaluated by expression of fluorescent marker.
Forma stericycle i160
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FORMA STERICYCLE i160 is a laboratory incubator designed for cell culture applications. It features a temperature-controlled chamber and precise control of environmental conditions to support the growth and maintenance of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mycoalerttm mycoplasma detection kit, by Lonza (1 mentions)
A375 cells, by American Type Culture Collection (1 mentions)
Mycophenolic acid, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by HiMedia (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
5 protocols using forma stericycle i160
1
Routine Cell Culture and Transfection
2
HEK293 Cell Culture and Mycoplasma Testing
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HEK293 cell cultures (from ATCC.org ) were incubated at 37°C, 5% CO2, 95% humidity in incubators (Thermo Fisher, FORMA STERI-CYCLE i160) in DMEM medium (Gibco,1199506) with 10% FBS (Gibco,1009914) and 1% penicillin streptomycin (Gibco, 15140122). The identity of the cell lines was not authenticated, and mycoplasma was not detected. Cells were split to 50% density and transfected with Lipofectamin (Invitrogen, 11668–019) 12 h later. Expression for the transfected constructs was evaluated by expression of fluorescent marker. All cultures tested negative for mycoplasma, which we tested by collecting 1 ml of DMEM cultured over 24 h with HEK293 cells, and centrifuge at 12,000 rpm for 1 min. Then, do the PCR as following primers: MGSO-TGCACCATCTGTCACTCTGTTAACCTC, GPO-3-GGGAGCAAACAGGATTAGATACCCT.
3
Adipocyte Differentiation of 3T3-L1 Cells
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Cell culture and differentiation of the adipocyte cell line followed the method
used by Kim and Jang (2014) (link) with some
modifications. In brief, the 3T3-L1 mouse embryo fibroblasts were obtained from
the Korean Cell Line Bank (Seoul, Korea). Growth media for the cell culture was
composed of 89% DMEM, 10% FBS and 1% Antibiotic-antimycotic
(%, v/v). Cells were cultured in the incubator (ThermoFisher Scientific,
FORMA STERICYCLE i160, Langenselbold, Germany) under 5% CO2 at
37°C. After the 3T3-L1 culture reached >90% confluence (Day
2), growth media supplemented with 0.5 mM IBMX, 1 μM DEX, and 1.7
μM insulin solution was applied. From Day 5 to Day 7, differentiation
media (containing 1 μM DEX and 1.7 μM Insulin in growth media) was
added to the 3T3-L1 cells. Finally, on Day 8 and Day 9, only 1.7 μM
insulin in growth media was added to the cell culture until the analysis.
used by Kim and Jang (2014) (link) with some
modifications. In brief, the 3T3-L1 mouse embryo fibroblasts were obtained from
the Korean Cell Line Bank (Seoul, Korea). Growth media for the cell culture was
composed of 89% DMEM, 10% FBS and 1% Antibiotic-antimycotic
(%, v/v). Cells were cultured in the incubator (ThermoFisher Scientific,
FORMA STERICYCLE i160, Langenselbold, Germany) under 5% CO2 at
37°C. After the 3T3-L1 culture reached >90% confluence (Day
2), growth media supplemented with 0.5 mM IBMX, 1 μM DEX, and 1.7
μM insulin solution was applied. From Day 5 to Day 7, differentiation
media (containing 1 μM DEX and 1.7 μM Insulin in growth media) was
added to the 3T3-L1 cells. Finally, on Day 8 and Day 9, only 1.7 μM
insulin in growth media was added to the cell culture until the analysis.
4
Cytotoxicity Evaluation of Human Cell Lines
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The cytotoxic assays were performed on three human cell lines: Hs27 derived from normal cells and two cell lines with different stages of melanoma progression. The human foreskin fibroblast Hs27 line was kindly provided by the Jagiellonian University Medical College in Krakow. WM115 cell line was obtained from the ESTDAB Melanoma Cell Bank (Tübingen; Germany). Human malignant melanoma A375 cells were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA).
Hs27 cells were grown in DMEM high glucose medium (Gibco, 41965–039, Paisley, UK) and the melanoma cells were cultured in RPMI 1640 medium (Gibco, 72400–054) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 10270–106), and 100 units mL−1 penicillin and 100 μg mL−1 streptomycin (Gibco, 151140–122). All cultures were maintained in a 37 °C humidified incubator with 5% CO2 (Forma Steri-Cycle i160, Thermo Scientific, Rockford, IL, USA).
When confluency was around 80–90%, cells were trypsinized using trypsin–EDTA solution (Sigma-Aldrich, T4049, St. Louis, MO, USA) and transferred into new cell culture dishes (TPP, Trasadingen, Switzerland) with a fresh media. All cell lines used in experiments were Mycoplasma-negative, which was determined by a MycoAlertTM Mycoplasma Detection Kit (Lonza, LT07–318, Walkersville, MD, USA).
Hs27 cells were grown in DMEM high glucose medium (Gibco, 41965–039, Paisley, UK) and the melanoma cells were cultured in RPMI 1640 medium (Gibco, 72400–054) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 10270–106), and 100 units mL−1 penicillin and 100 μg mL−1 streptomycin (Gibco, 151140–122). All cultures were maintained in a 37 °C humidified incubator with 5% CO2 (Forma Steri-Cycle i160, Thermo Scientific, Rockford, IL, USA).
When confluency was around 80–90%, cells were trypsinized using trypsin–EDTA solution (Sigma-Aldrich, T4049, St. Louis, MO, USA) and transferred into new cell culture dishes (TPP, Trasadingen, Switzerland) with a fresh media. All cell lines used in experiments were Mycoplasma-negative, which was determined by a MycoAlertTM Mycoplasma Detection Kit (Lonza, LT07–318, Walkersville, MD, USA).
5
DENV-2 Replicon Assay Protocol
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Baby Hamster Kidney (BHK-21) cells were procured from National Centre for Cell Science, Pune, India. Dulbecco's modified Eagle medium (Himedia; catalogue#AT149), FBS (Life Technologies; catalogue#10439-016), L-Glutamine (Sigma; catalogue#G7513), Penicillin-Streptomycin (Sigma; catalogue#P4333), mycophenolic acid (Sigma; catalogue#M5255) an inhibitor of DENV-RNA replication, were procured from the supplier (Sigma-Aldrich®). The cells were cultured and maintained at 37 °C with 5% CO 2 incubator (Thermo Scientific™ Forma™ Steri-Cycle i160).
The pRS424 plasmid containing frame work of non-structural genes of DENV 2 (NGCstrain) was a kind gift from Prof. Padmanabhan's laboratory (Georgetown University, Washington DC, USA). The pRS424 plasmid containing coding regions (RLuc-IRES-Neo r -DENV2-NS) were cloned downstream of SP6 promoter. The gene order of replicon is 5′ UTR, N terminal coding sequence of capsid (C), Renilla Luciferase reporter (Rluc) with translational termination codon and internal ribosome entry site (IRES) for 5′-cap-independent translation of the downstream ORF that encodes a poly protein precursor C terminus E-NS1-NS2A, NS2B, NS4A, NS4B, NS5 followed by 3′ UTR.
The pRS424 plasmid containing frame work of non-structural genes of DENV 2 (NGCstrain) was a kind gift from Prof. Padmanabhan's laboratory (Georgetown University, Washington DC, USA). The pRS424 plasmid containing coding regions (RLuc-IRES-Neo r -DENV2-NS) were cloned downstream of SP6 promoter. The gene order of replicon is 5′ UTR, N terminal coding sequence of capsid (C), Renilla Luciferase reporter (Rluc) with translational termination codon and internal ribosome entry site (IRES) for 5′-cap-independent translation of the downstream ORF that encodes a poly protein precursor C terminus E-NS1-NS2A, NS2B, NS4A, NS4B, NS5 followed by 3′ UTR.
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