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T25 flask

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The T25 flask is a laboratory equipment item used for the culturing of cells. It provides a standardized container with a surface area of approximately 25 square centimeters for the growth and maintenance of cell lines and other biological samples.

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Lab products found in correlation

Penicillin, by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) M avium jcm15430, by RIKEN BioResource Center (1 mentions) M intracellulare jcm6384, by RIKEN BioResource Center (1 mentions) Ham s f 12 nutrient mix, by Thermo Fisher Scientific (1 mentions) Lithium chloride precipitation solution, by Thermo Fisher Scientific (1 mentions) Dnase 1, by New England Biolabs (1 mentions) Rnase free dnase 1, by New England Biolabs (1 mentions) Phosphofructokinase activity colorimetric assay kit, by Merck Group (1 mentions) Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions)

6 protocols using t25 flask

1

Culturing and Cryopreserving Primary Cells

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Example 1

Cells from whole peripheral blood, whole umbilical cord blood mononuclear fraction and RBC fraction were cultured separately and allowed to adhere to the bottom of the T25 flask (Techno Plastic products TPP).

Culture was initially incubated in growth medium for four days. Medium changes were carried out twice weekly thereafter. Growth medium used was Low-glucose Dulbecco's Modified Eagle's medium LG DMEM (Gibco) with GlutaMAX™ and supplemented with 10% heat inactivated fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) incubated at 37° C. and 5% CO2 in a humidified atmosphere using the incubator (SHEL LAB, USA).

At later stages only half the medium was weekly changed. Cells were usually passaged upon reaching 80% to 90% confluency using a scraper or cold shock treatment (˜1 h at 4-8° C.). Primary culture cells reached confluency in 3-4 weeks. SMS cells were able to proliferate into a multilayer (at least three layers) (see, e.g., FIG. 1C).

Lower layer cells became attached more strongly to the surface of the flask through a layer of extra cellular matrix (ECM). Cells were cryopreseved at different passages in cryopreserving medium (60% growth medium, 30% added FBS, 10% DMSO) and successfully revived in the same growth medium.

US10041037B2. Small mobile stem cells (SMS) and uses thereof (2018-08-07). SMSBIOTECH, INC. [US]. Inventors: Abdulkader Rahmo [US].
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2

Culturing and Cryopreserving Primary Cells

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Example 1

Cells from whole peripheral blood, whole umbilical cord blood mononuclear fraction and RBC fraction were cultured separately and allowed to adhere to the bottom of the T25 flask (Techno Plastic products TPP).

Culture was initially incubated in growth medium for four days. Medium changes were carried out twice weekly thereafter. Growth medium used was Low-glucose Dulbecco's Modified Eagle's medium LG DMEM (Gibco) with GlutaMAX™ and supplemented with 10% heat inactivated fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) incubated at 37° C. and 5% CO2 in a humidified atmosphere using the incubator (SHEL LAB, USA).

At later stages only half the medium was weekly changed. Cells were usually passaged upon reaching 80% to 90% confluency using a scraper or cold shock treatment (˜1 h at 4-8° C.). Primary culture cells reached confluency in 3-4 weeks. SMS cells were able to proliferate into a multilayer (at least three layers) (see, e.g., FIG. 1C).

Lower layer cells became attached more strongly to the surface of the flask through a layer of extra cellular matrix (ECM). Cells were cryopreseved at different passages in cryopreserving medium (60% growth medium, 30% added FBS, 10% DMSO) and successfully revived in the same growth medium.

US11345886B2. Small mobile stem cells (SMS) and uses thereof (2022-05-31). SMSBIOTECH, INC. [US]. Inventors: Abdulkader Rahmo [US].
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3

Culturing Mycobacterium avium and intracellulare

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M. avium JCM15430 and M. intracellulare JCM6384 were obtained from the Riken BioResource Research Center (Ibaraki, Japan). Both strains were stored in 20% glycerol at -80°C. Frozen stock culture (500 µL, approximately 5.0 × 10 8 colony-forming unit (CFU)/ mL) was inoculated into Middlebrook 7H9 broth (10 mL) in a T-25 flask (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and cultured under static conditions at 37°C for 14 days (up to approximately 1.0 × 10 9 CFU/mL).
Yagi A., Yamazaki H., Terahara T., Yang T., Hamamoto H., Imada C., Tomoda H, & Uchida R. (2021). Development of an in vivo-mimic silkworm infection model with Mycobacterium avium complex. Drug discoveries & therapeutics, 14(6).
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4

Immortalized Microglial Response to LPS

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BV2 microglia are derived raf/myc-immortalized murine neonatal microglia. They were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 nutrient mix supplemented with 10% fetal bovine serum and antibiotics (penicillin 100U/ml, streptomycin 100ug/ml; all from Life Technologies Corp., Grand Island, NY). Cells were kept at 37°C in a humidified atmosphere of air and 5% CO2 and propagated in T25 flasks (Techno Plastic Products AG, Trasadingen, Switzerland) in media with or without 100mM EtOH in sealable plastic bags as described above to avoid EtOH evaporation. They were split every 2–3 days and subjected to EWD after 10 days. During EWD, cells were seeded in 24 well plates at densities of 5×105 cells/well, allowed to adhere overnight and then challenged with 1μg/mL LPS for 24h. Culture media was collected and assessed for inflammatory mediators as described above. Cell viability was measured using resazurin salt fluorescence (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt; Sigma Aldrich Co. LCC., St. Louis, MO) and normalized to percentage control (no LPS group).
Lutz J.A., Carter M., Fields L., Barron S, & Littleton J.M. (2015). Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal. Alcoholism, clinical and experimental research, 39(5), 827-835.
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5

Isolating High-Quality RNA for TERRA Analysis

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To perform this method, it is important to isolate high-quality RNA. We used phenol chloroform extraction to preserve RNA integrity. Cells grown in T-25 flasks (Techno Plastic Products, c Trassadigen, Switzerland) were centrifuged. Cell pellets were suspended in 1 mL of TRIzol™ Reagent (ThermoFisher), and 200 µL of chloroform was added. Samples were vortexed and centrifuged at 12,000× g for 15 min at 4 °C. The aqueous phase was collected, and RNA was precipitated with 1 volume of isopropanol. The pellets were washed with 70% ethanol, and RNA was dissolved in 50 µL H2O.
Since TERRA is present in small amounts in human telomerase-positive cell lines, it was essential to completely remove any DNA contamination using an extensive DNase I treatment. For this, 40 µg RNA (digestion was incomplete if a higher amount of RNA was used) were treated with 2 µL RNase-free DNase I (New England Biolabs, Evry, France ) in a 50 µL volume, including 5 µL 10× DNase I Reaction buffer, 1 µL RNase Inhibitor, Murine (New England Biolabs) for 30 min at 37 °C. RNA was precipitated with Lithium Chloride Precipitation Solution (Invitrogen), according to the manufacturer’s protocol, resuspended in 30 µL H2O, and quantified by UV spectrometry (Nanodrop, Thermo Fisher, Illkirch, France ).
Le Berre G., Hossard V., Riou J.F, & Guieysse-Peugeot A.L. (2020). AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites. Biomolecules, 10(6), 827.
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6

Quantification of Phosphofructokinase Activity

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Phosphofructokinase measurements were done according to the manufacturer’s datasheet (Phosphofructokinase Activity Colorimetric Assay Kit, Sigma, Switzerland). SH-SY5Y cells were seeded in T25 flasks (Techno Plastic Products AG (TPP), Trasadingen, Switzerland) at a density of 3 × 106 cells per flask and treated as previously described above. Briefly, the protein was extracted by scraping off cells in 1 mL HBSS and centrifuged at 13.200×g for 10 min. The buffer was removed and the pellet was dissolved in 200 µL cold PFK assay buffer and centrifuged at 13.200×g for 10 min. The pellet was solubilized in 60 µL cold PFK buffer and 50 µL were transferred to a 96- well plate and a master mix consisting of PFK Assay Buffer, PFK Enzyme Mix, PFK Developer, ATP and PFK substrate was added. The absorbance (450 nm) was measured using a Synergy H1 multi-mode reader. The data were compared with the NADH standard curve ranging from [2 to 10 nmol]/well and values were normalized to 1 mg protein. The protein amount of each sample was determined with Pierce™ 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Switzerland).
Ducray A.D., Felser A., Zielinski J., Bittner A., Bürgi J.V., Nuoffer J.M., Frenz M, & Mevissen M. (2017). Effects of silica nanoparticle exposure on mitochondrial function during neuronal differentiation. Journal of Nanobiotechnology, 15, 49.
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