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Co2 independent media

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CO2-independent media is a type of culture media used in cell culture applications. It is designed to maintain cell growth and viability without the need for a CO2-enriched environment. The media is formulated to buffer pH changes, allowing cells to proliferate in standard incubator conditions.

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Lab products found in correlation

Penicillin, by Thermo Fisher Scientific (4 mentions) Glass bottom culture dishes, by MatTek (4 mentions) Bovine serum albumin (bsa), by Merck Group (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions) Deltavision softworx software, by Cytiva (4 mentions) Dulbecco s modified eagle medium, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Nis elements software, by Nikon (3 mentions) Coolsnap hq2, by Cytiva (3 mentions) 8 well glass bottomed chambers, by Ibidi (3 mentions) Deltavision core deconvolution microscope, by Cytiva (3 mentions) Sucrose, by Merck Group (2 mentions) Glosensor camp assay, by Promega (2 mentions) Glosensor camp reagent, by Thermo Fisher Scientific (2 mentions) Plan fluor objective 1.3na, by Nikon (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) 75 cm2 culturing flask, by Corning (2 mentions) Epower 0483e7, by Thermo Fisher Scientific (2 mentions)

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Leibovitz CO2-independent media (4 citations)

55 protocols using co2 independent media

1

GloSensor cAMP Assay for Cell-Based Analysis

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Example 10

GloSensor cAMP assay

The GloSensor cAMP assay was conducted per manufacturer's protocol (Promega). In brief, ChoK1 human GLP-1R/GIPR-expressing cells were seeded at 15,000 cells per well in a 96 well plate and incubated for 16 hours in a humidified 37° C., 5% CO2 incubator. Cells were transiently transfected with 100 ng pGloSensor −22F cAMP plasmid using FuGENE HD for 24 hours. Cells were then equilibrated with 0.1% BSA, 2% GloSensor cAMP reagent in CO2 independent media (Invitrogen) for 2 hours at room temperature. Cells were preincubated for 15 min with or without 0.43 M sucrose (Sigma). Basal luminescence measurements were taken for 10 minutes prior to addition of 1248 in conditions with or without sucrose. Immediately following addition of 1248, kinetic luminescence was measured using an integration time of 0.1-1 second every 60 seconds. Data were analyzed using GraphPad Prism software and presented in FIG. 26.

US10905772B2. Method of treating or ameliorating metabolic disorders using GLP-1 receptor agonists conjugated to antagonists for gastric inhibitory peptide receptor (GIPR) (2021-02-02). AMGEN INC. [US]. Inventors: Yuan Cheng [US], Chawita Netirojjanakul [US], Jerry Ryan Holder [US], Bin Wu [US], James R. Falsey [US], Bradley J. Herberich [US], Kelvin Sham [US], Leslie P. Miranda [US], Shu-Chen Lu [US], Murielle M. Veniant-Ellison [US], Shanaka Stanislaus [US], Junming Yie [US], Jing Xu [US].
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2

Live-cell Imaging of Kinetochore Dynamics

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Cells were imaged in CO2-independent media (Invitrogen) at 37°C. All images except laser microsurgery were acquired on a Nikon eclipse microscope equipped with a CCD camera (Clara, Andor) using a 40x Plan Fluor objective 1.3NA (Nikon) and appropriate fluorescence filters. Images of 3xGFP-CENP-A cell lines were acquired every 8 seconds using 3 (HeLa) or 5 (hTERT RPE-1 and Figure S3C) z sections at 0.7 μm intervals. Where indicated, cells were imaged at 4s interval using a single plane focus. mNeonGreen-PICH cells were imaged every 60s at 4 z-sections at 1 μm intervals. The laser microsurgery was conducted as described previously (Pereira et al., 2009 (link)) and is detailed in the Supplemental Experimental Procedures. An extended description of the analysis of the time-lapse movies and kinetochore motion is also included in the Supplemental Experimental Procedures.
Su K.C., Barry Z., Schweizer N., Maiato H., Bathe M, & Cheeseman I.M. (2016). A Regulatory Switch Alters Chromosome Motions at the Metaphase to Anaphase Transition. Cell reports, 17(7), 1728-1738.
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3

Live-cell Imaging of Cell Migration

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HeLa Kyoto cells stably expressing AcGFP-LAP2β and H2B-mCherry were transfected with siRNA and cultured for 50 h before recording. Prior to start of recording, the culture medium was changed to phenol-red free and CO2 independent medium (Invitrogen). Images were acquired as 4 z-planes 3 μm apart every 3 min at 37 °C using a Nikon TE2000 microscope equipped with a Plan Fluor × 60/1.4 DIC H Lens (Nikon), a PerkinElmer ERS Spinning disk system, a Digital CCD C4742-80-12AG camera (Hamamatsu), and controlled by Volocity 6.0.1 software. Cell images were projected with z-stacks and analysed by ImageJ. For analysis of cell migration, MDA-MB-231 cells were transfected with either control or PPP1R12A-siRNA using Lipofectamine 2000 (Invitrogen). The following day the cells were trypsinized and seeded in CO2 independent media (Invitrogen) in 24 well MatTek dishes (MatTek Corporation). Six to eight hours after seeding the cells were imaged for 20 h using phase contrast microscopy using an adapted Zeiss LSM510 microscope. Cell tracking was performed using the Manual Tracking plugin in Fiji software.
Takaki T., Montagner M., Serres M.P., Le Berre M., Russell M., Collinson L., Szuhai K., Howell M., Boulton S.J., Sahai E, & Petronczki M. (2017). Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability. Nature Communications, 8, 16013.
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4

FACS-based MLV/luc Fusion Assay

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To investigate MLV/luc fusion to Rat2 target cells, a FACS based assay29 (link) was utilized with some modification. Briefly, untreated or laser-exposed MLV/luc particles containing S15-BlaM fusion protein was used to infect target cells. After incubating 2 hours at 37 °C, cells were washed with CO2-independent media (Invitrogen) and incubated in CCF2 (fluorogenic substrate of β-lactamase)-containing media at 18 °C over night, washed, fixed with 4% PFA. The number of β-lactamase positive cells was determined by FACS analysis using a LSRII flow cytometer (BD).
Nazari M., Xi M., Lerch S., Alizadeh M.H., Ettinger C., Akiyama H., Gillespie C., Gummuluru S., Erramilli S, & Reinhard B.M. (2017). Plasmonic Enhancement of Selective Photonic Virus Inactivation. Scientific Reports, 7, 11951.
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5

Adoptive transfer of OTII T cells

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Magnetically separated WT or miR-155−/− OTII CD4+ T cells, were labeled with 10 μg/mL CFSE (life technologies, CA, USA) in PBS for 20 min at 37°C as recommended by the manufacturer (10 × 106 cells/ml). After labeling, 2–5 × 106 cells/mouse were injected intravenously into either WT, miR-155−/− or CD45.1 mice. For multiphoton imaging, 2–5 × 106 cells/mouse were labeled with 5 μM Cell Tracker Red (CMTPX, Invitrogen, Paisley, Scotland, UK) in CO2-independent media (Invitrogen, Paisley, Scotland, UK) for 40 min at 37°C, and injected i.v. into CD11c-YFP mice. For immunization experiments with HEL-OVA, OTII T cells, and MD4 B cells were prepared and injected as described before (26 (link)).
Goncalves-Alves E., Saferding V., Schliehe C., Benson R., Kurowska-Stolarska M., Brunner J.S., Puchner A., Podesser B.K., Smolen J.S., Redlich K., Bonelli M., Brewer J., Bergthaler A., Steiner G, & Blüml S. (2019). MicroRNA-155 Controls T Helper Cell Activation During Viral Infection. Frontiers in Immunology, 10, 1367.
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6

Live-cell Imaging of Kinetochore Dynamics

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Cells were imaged in CO2-independent media (Invitrogen) at 37°C. All images except laser microsurgery were acquired on a Nikon eclipse microscope equipped with a CCD camera (Clara, Andor) using a 40x Plan Fluor objective 1.3NA (Nikon) and appropriate fluorescence filters. Images of 3xGFP-CENP-A cell lines were acquired every 8 seconds using 3 (HeLa) or 5 (hTERT RPE-1 and Figure S3C) z sections at 0.7 μm intervals. Where indicated, cells were imaged at 4s interval using a single plane focus. mNeonGreen-PICH cells were imaged every 60s at 4 z-sections at 1 μm intervals. The laser microsurgery was conducted as described previously (Pereira et al., 2009 (link)) and is detailed in the Supplemental Experimental Procedures. An extended description of the analysis of the time-lapse movies and kinetochore motion is also included in the Supplemental Experimental Procedures.
Su K.C., Barry Z., Schweizer N., Maiato H., Bathe M, & Cheeseman I.M. (2016). A Regulatory Switch Alters Chromosome Motions at the Metaphase to Anaphase Transition. Cell reports, 17(7), 1728-1738.
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7

Src Activity Measurement via FRET Biosensor

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Src activity was determined using a FRET based Src biosensor as previously described [27 (link), 28 (link)] with minor modifications. The Src biosensor consists of a CFP-YPet FRET pair at either end of a flexible linker domain containing a Src kinase peptide (WMEDYDYVHLQG, from the known Src target p130cas[29 (link), 30 (link)]). NIH 3T3 cells were transiently transfected with 3µg β2AR, 2 µg Src biosensor, and 2 µg of mCherry-tagged Src mutants. Fibroblasts were plated onto poly-d-lysine coated dishes and starved for 36 hours in MEM containing 0.5% FBS. Prior to imaging, media was changed to CO2-independent media (Invitrogen) and the cells were imaged on a Deltavision Elite microscope using a 60× oil immersion objective with a 1.42 NA and an Evolve back-thinned EM-CCD camera. Images were acquired every 2 min using CFP-YFP-mCherry filter sets. Excitation of the samples was performed by an InsightSSI 7 channel solid-state illumination source. Wortmannin (50 nM) or PP2 (10 µM) were added 30 min prior to stimulation with ISO (10 µM) or PDGF (50 nM). Vehicle or agonist was added after the third time point. Acquired images were analyzed using ImageJ. Normalized emission ratios (CFP/FRET signal) are reported.
Watson L.J., Alexander K.M., Mohan M.L., Bowman A.L., Mangmool S., Xiao K., Naga Prasad S.V, & Rockman H.A. (2016). Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor mediated EGFR transactivation. Cellular signalling, 28(10), 1580-1592.
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8

Isolation and Differentiation of Fetal Human RPE

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Permission to work with human RPE was obtained from the Ethics Committee of the University of Bonn. Two pairs of fetal human eyes at 19th and 20th week of gestation were obtained from Advanced Bioscience Resources. The tissues were transported in CO2-independent media (Invitrogen) supplemented with 5% normal calf serum and 100 IU penicillin/100 μg streptomycin on ice and were processed within 48 hr postenucleation. RPE cells were isolated, expanded in low calcium media, and subsequently differentiated on uncoated 10-μm-thick polyester Transwell (PET) inserts (Corning Life Sciences catalog number 3470) according to a prior protocol (Hu and Bok, 2001 (link)).
Stanzel B.V., Liu Z., Somboonthanakij S., Wongsawad W., Brinken R., Eter N., Corneo B., Holz F.G., Temple S., Stern J.H, & Blenkinsop T.A. (2014). Human RPE Stem Cells Grown into Polarized RPE Monolayers on a Polyester Matrix Are Maintained after Grafting into Rabbit Subretinal Space. Stem Cell Reports, 2(1), 64-77.
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9

Extraction of Cells from lrECM Gels for AFM

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Single cells and multicellular structures were extracted from the lrECM gels for AFM study with an adapted version of previously described acinus-extraction method [38] (link). The lrECM gels were quickly washed with PBS and then mechanically detached from the culture well. To dissolve the matrix, embedded gels were soaked in a iced PBS-EDTA mixture (0.5 M EDTA pH 8.0 from Invitrogen diluted to 5.5 µM final concentration in PBS) for 10 minutes before being placed in a 1.5 mL tube with excess PBS-EDTA for an additional 25 minutes. The resulting mixture was gently centrifuged at 100–200g (single cells 3–5 minutes; acini ∼10 s) and the supernatant was aspirated away. Cells/acini were resuspended in CO2-independent media (Invitrogen) with 10% fetal bovine serum and 1× penicillin–streptomycin and plated on a poly-L-lysine-coated (MW>300,000, P5899 Sigma-Aldrich) cover slip for AFM experiments. Poly-L-lysine coatings were used to allow samples to electrostatically attach without activating cell adhesion machinery on the surface.
Venugopalan G., Camarillo D.B., Webster K.D., Reber C.D., Sethian J.A., Weaver V.M., Fletcher D.A., El-Samad H, & Rycroft C.H. (2014). Multicellular Architecture of Malignant Breast Epithelia Influences Mechanics. PLoS ONE, 9(8), e101955.
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10

Time-lapse Microscopy of NIH3T3 Cell Migration

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NIH3T3 cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco Invitrogen), with 10% fetal bovine serum (FBS) (Gibco Invitrogen) and 100 units/ml penicillin (Gibco Invitrogen) in 75-cm2 culturing flask (Corning) at 37 °C and 5% CO2 culture incubator. Before experimentation, confluent cells were detached from the flask through treatment with 0.05% Trypsin-EDTA (Gibco Invitrogen) and seeded onto the patterned substrate in sterile polystyrene well plates with CO2-independent media (Gibco Invitrogen), 10% FBS, 100 units/ml penicillin, and 1% GlutaMAX (Gibco Invitrogen) and placed on an inverted fluorescence microscope (IX71, Olympus) that was covered by a chamber to maintain temperature and humidity. Phase-contrast images were taken every 5 minutes with a digital CCD camera (Retiga 2000R cooled, Qimaging) for about 2–3 days.
Jeon H., Koo S., Reese W.M., Loskill P., Grigoropoulos C.P, & Healy K.E. (2015). Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces. Nature materials, 14(9), 918-923.
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