DBTRG .05MG. Cells were grown as a monolayer in 25 cm2 tissue culture flasks (Nunclon™, Denmark) at 37°C in an atmosphere of 5% CO2 in RPMI-1640 medium for DBTRG .05MG supplemented with 10% fetal calf serum and 1%antibiotics. Three additional supplements were needed, 2 mM Glutamine, 1% HT, and 1 mM Sodium Pyruvate. Cells were maintained at 37 °C in 5%CO2 atmosphere. Further maintenance and subculturing of cells were done according to supplier’s protocol.
25 cm2 tissue culture flask
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
The 25 cm2 tissue culture flasks are a standard laboratory equipment used for in vitro cell culture. The flasks provide a controlled environment for the growth and maintenance of cells, tissues, or microorganisms. They feature a flat surface area of 25 square centimeters to accommodate cell attachment and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin g streptomycin, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
24 well culture plate, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem high glucose, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Maltose, by Merck Group (1 mentions)
Dulbecco s modified eagle s minimal essential medium dmem, by Merck Group (1 mentions)
Cellobiose, by Merck Group (1 mentions)
Cysteine, by Merck Group (1 mentions)
Penicillin g streptomycin amphotericin b, by Thermo Fisher Scientific (1 mentions)
Aminopterin, by Merck Group (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Polyethylene glycol, by Merck Group (1 mentions)
Sp2 0 cell, by American Type Culture Collection (1 mentions)
12 protocols using 25 cm2 tissue culture flask
1
DBTRG .05MG Cell Culture Protocol
2
Co-culture of HT-29 Cells with F. prausnitzii
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The human colon carcinoma cell line HT-29 (ATCC HTB-38) was grown in Dulbecco’s Modified Eagle’s minimal essential medium with 4.5 g/L glucose (DMEM) (Sigma-Aldrich), supplemented with 10% (w/v) heat-inactivated fetal calf serum (FCS) (GibcoBRL, Eragny, France), 4 mM L-glutamine, and penicillin G/streptomycin (5000 IU/mL, 5000 µg/mL) (Sigma-Aldrich). Cultures were incubated in 25-cm2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37ºC in a 10% (v/v) CO2 atmosphere until confluence.
For co-culture experiments, HT-29 cells were seeded in 24-well culture plates (Nunc) in DMEM supplemented with 10% heat-inactivated FCS-1% glutamine at 37°C in a 10% CO2-air atmosphere. Culture medium was changed every day. Experiments began on day 7 after seeding, when cells were at confluence (approx. 1.83 × 106 cells/well). On day 6, 24 h before co-culture with F. prausnitzii SN, the culture medium was changed to one with 5% heat-inactivated FCS and 1% glutamine. The day of the co-culture, either SN or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 500 µl. Cells were stimulated simultaneously with recombinant human TNF-α (5 ng/ml; Peprotech, NJ, USA) for 6 h at 37°C in 10% CO2. All samples were analyzed in triplicate.
For co-culture experiments, HT-29 cells were seeded in 24-well culture plates (Nunc) in DMEM supplemented with 10% heat-inactivated FCS-1% glutamine at 37°C in a 10% CO2-air atmosphere. Culture medium was changed every day. Experiments began on day 7 after seeding, when cells were at confluence (approx. 1.83 × 106 cells/well). On day 6, 24 h before co-culture with F. prausnitzii SN, the culture medium was changed to one with 5% heat-inactivated FCS and 1% glutamine. The day of the co-culture, either SN or LYBHI medium was added at a concentration of 10% (v/v) in a total volume of 500 µl. Cells were stimulated simultaneously with recombinant human TNF-α (5 ng/ml; Peprotech, NJ, USA) for 6 h at 37°C in 10% CO2. All samples were analyzed in triplicate.
3
Culturing BUVECs and Infecting with Sporozoites
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BUVECs were cultured in 25 cm2 tissue culture flasks (Nunc) at 37°C and 5% CO2 atmosphere until confluency and infected with 5 × 105 freshly excysted sporozoites. Cell culture medium was changed 1 day after infection and thereafter every third day. Infection rates were determined at 1 day p.i. microscopically.
4
Isolation and Expansion of Human UCB-MSCs
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Collection, isolation and expansion of human UCB-MSCs was performed as previously described (18 (link)-20 (link)). The mononuclear cells (MNCs) fraction was separated by Ficoll-Hypaque low-density [<1.077 g/ml (Cedar Lane, Canada)] gradient followed by ammonium chloride lysis of red blood cells. After twice washing by phosphate-buffered saline (PBS; Gibco, USA), the collected MNCs were re-suspended in high glucose-Dulbecco’s modified eagle medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), L-glutamine (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco). MSCs were cultured in 25 cm2 tissue culture flasks (Nunc, USA) in a humidified atmosphere of 95% air with 5% CO2 at 37 °C.
5
BMSC Isolation and Transplantation into Rat Fetus
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BMSCs were isolated from the bone marrow of 4-week-old Wister rats and were cultured in Dulbecco’s modified Eagle’s Medium: nutrient mixture F-12 (DMEM/F12; HyClone, USA) supplemented with 10% fetal bovine serum (FBS; Corning Life Sciences/Cellgro, USA) in 25-cm2 tissue culture flasks (Nunc, USA). Primary isolated BMSCs were defined as P0. At confluency, cells were passaged (1:2) with fresh medium, and P3–P4 were used for transplantation. To visualize the BMSCs after transplantation into rat fetus, cells were transfected with enhanced green fluorescent protein (EGFP)-expressing adeno-5 vector (Ad-GEP, 100 PFU/cell; Hanbio, China) before transplantation (24 h). The Ad-GFP-transfected BMSCs were trypsinized, centrifuged, and resuspended in PBS before in utero injection. GFP+ve BMSCs were counted and adjusted with PBS to yield concentrations ranging from 2 × 109 to 3 × 109 cells/μL.
6
Growth of Faecalibacterium prausnitzii Strains
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The reference strains A2-165 (DSM 17677; Duncan et al., 2002 (link)), L2/6 (Barcenilla et al., 2000 (link)) and M21/2 (Louis et al., 2004 (link)) and the F. prausnitzii isolated strains (Table 1 ) were grown at 37°C in YBHI medium supplemented with cellobiose (1 mg/ml; Sigma), maltose (1 mg/ml; Sigma), and cysteine (0.5 mg/ml; Sigma) in an anaerobic chamber filled with N2 = 90%, CO2 = 5% and H2 = 5%.
HT-29 (ATCC HTB-38) (LGC-Standars) cell line was grown in Dulbecco's Modified Eagle's minimal essential medium (DMEM) (Sigma-Aldrich) supplemented with 10% (w/v) heat-inactivated fetal bovine serum (FBS) (GibcoBRL, Eragny, France) and with penicillin G/ streptomycin (5,000 IU/mL, 5,000 μg/mL) (Sigma-Aldrich). Cultures were incubated in 25 cm2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37°C in a 5% (v/v) CO2 atmosphere until confluence.
HT-29 (ATCC HTB-38) (LGC-Standars) cell line was grown in Dulbecco's Modified Eagle's minimal essential medium (DMEM) (Sigma-Aldrich) supplemented with 10% (w/v) heat-inactivated fetal bovine serum (FBS) (GibcoBRL, Eragny, France) and with penicillin G/ streptomycin (5,000 IU/mL, 5,000 μg/mL) (Sigma-Aldrich). Cultures were incubated in 25 cm2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37°C in a 5% (v/v) CO2 atmosphere until confluence.
7
Adenovirus Infection Kinetics in HEK-293 Cells
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The HEK-293 cell monolayer grown in 25 cm2 tissue culture flasks (Nunc) was infected separately with the recombinant adenoviruses or non-recombinant replication-defective human adenovirus 5 (dAd5, available at IVRI, Bangalore) at an MOI of 0.1. The viruses were allowed to adsorb for 1 h and then the monolayer was washed twice with PBS. The infected cells were harvested at 0 h (immediately after adsorption) and at every 12 h intervals post infection up to 5 days and frozen at −80°C. Titer of each sample was determined in duplicate and mean value was used to study the growth kinetics of the adenoviruses.
8
Culturing HT29 Colon Adenocarcinoma Cells
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The human colon adenocarcinoma cell line HT29 was grown in DMEM (Dulbecco’s modified Eagle’s medium) (GibcoBRL, Eragny, France) supplemented with 10% (w/v) fetal bovine serum (GibcoBRL) and with one of: Penicillin G/streptomycin/Amphotericin B (10,000 IU/mL, 10,000 μg/mL, 25 μg/mL) (GibcoBRL, Grand Island, NY, USA). Cultures were incubated in 25 cm2 tissue culture flasks (Nunc, Roskilde, Denmark) at 37 °C in a 5% (v/v) CO2 atmosphere.
9
Monoclonal Antibody Production from Hybridoma Cells
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To produce hybridoma cell-secreted monoclonal antibodies (mAbs), we used a cell fusion technique according to an established protocol [30 (link),31 (link)]. Mouse myeloma cells (SP2/0 cell; ATCC #CRL 1581) were fused with spleen cells from the donor mice using 50% polyethylene glycol (Sigma Chemical Co.). After incubation with hypoxanthine, aminopterin, and thymidine media (Sigma Chemical Co.), supernatants of hybridoma cell culture were screened by ELISA using the recombinant ZIKV NS1 or E proteins (1 μg/ml) as an antigen. After subcloning with limiting dilutions, selected hybridoma colonies were transferred into 25 cm2 tissue culture flasks (Nunc, Roskilde, Denmark) with RPMI-1640 medium (Gibco/BRL) containing 10% FBS (Gibco/BRL).
10
PC12 Cell Culture Protocol
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PC12 cells (Health Science Research Resources Bank, Osaka, Japan) were seeded in 25 cm2 tissue culture flasks (Nunc, Roskilde, Denmark) and cultured in Dulbecco Eagle’s minimum essential medium (DMEM, Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated horse serum (Invitrogen, Carlsbad, CA, USA) and 5% fetal bovine serum (FBS, Gibco-BRL, Grand Island, NY, USA), 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C.
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