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Co2 independent medium

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CO2-independent medium is a type of cell culture medium formulated to support cell growth and maintenance in the absence of a supplemental CO2 environment. It is designed to maintain the appropriate pH and nutrient levels for cell lines that do not require a CO2-enriched atmosphere.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions) Dnase 1, by Merck Group (7 mentions) Hyaluronidase, by Merck Group (7 mentions) L glutamine, by Thermo Fisher Scientific (7 mentions) Glutamax, by Thermo Fisher Scientific (7 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions) Dnase, by Roche (6 mentions) Liberase tl, by Roche (5 mentions) Glass bottom culture dishes, by MatTek (4 mentions) Axio observer z1 microscope, by Zeiss (4 mentions) Penicillin, by Thermo Fisher Scientific (3 mentions) Pulsed nitrogen laser, by Spectra-Physics (3 mentions) 35 mm glass bottom dishes, by MatTek (3 mentions) Dispase, by Roche (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) Deae dextran, by Merck Group (3 mentions) Glosensor camp reagent, by Promega (3 mentions) Inverted phase contrast microscope, by World Precision Instruments (3 mentions)

Close spelling variants of this product

CO2-independent culture medium Phenol red-free CO2-independent medium Phenol-red free and CO2 independent medium CO2-independent imaging medium L-15 CO2-independent medium Complete CO2-independent medium

206 protocols using co2 independent medium

1

HIV Viral Fusion Assay in DCs

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Viral fusion was analyzed as described [29 (link)]. Briefly, day 5 DCs were plated into a 96-well plate in triplicates (1.5x105 cells/100μl) in RPMI in presence of 10mM Hepes (Life Technologies) and 2mg/ml DEAE-Dextran (Sigma-Aldrich). Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing Blam-Vpr. After 3 hours, cells were washed 2 times in CO2-independent medium (Life Technologies), re-suspended in CO2-independent medium containing 10% FCS and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G Loading Kit with CCF2-AM, Life Technologies). After 2h incubation at room temperature (dark) cells were washed 2 times in CO2-independent medium, fixed in 4% paraformaldehyde for 30 min and respective wells were pooled for flow cytometric analysis.
Posch W., Steger M., Knackmuss U., Blatzer M., Baldauf H.M., Doppler W., White T.E., Hörtnagl P., Diaz-Griffero F., Lass-Flörl C., Hackl H., Moris A., Keppler O.T, & Wilflingseder D. (2015). Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells. PLoS Pathogens, 11(6), e1005005.
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2

Cortical Cell Infection by HSV-1

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At day of in vitro 6 (DIV6), the primary cortical cells were incubated with CO2-independent medium (Gibco) containing 0.1% BSA for 20 min at room temperature on a rocking platform. To prepare the inoculum, HSV-1 stocks were diluted with CO2-independent medium (Gibco) containing 0.1% (w/v) BSA to a multiplicity of infection (MOI) of 10 pfu/cell (corresponding to 2.8 × 106 pfu/mL), and added to the cells for 30 min on a rocking platform. After infection, cells were washed with starvation medium once and incubated with starvation medium at 37°C for 20 h.
Hensel N., Raker V., Förthmann B., Buch A., Sodeik B., Pich A, & Claus P. (2020). The Proteome and Secretome of Cortical Brain Cells Infected With Herpes Simplex Virus. Frontiers in Neurology, 11, 844.
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3

Live-cell Imaging of Mitotic Progression

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Cells were plated on an 8 well 15 μ-Slide (Ibidi, Martinsried, Germany). CENP-P was depleted as previously described for 48 h prior to imaging. For asynchronous cells, FlpIn T-REx HeLa cells were transferred into CO2 Independent Medium (Gibco) 16 h before timelapse. Two hours before imaging SiR-Hoechst DNA dye (Spirochrome) was added. Timelapse demonstrating recovery from nocodazole treatment utilized U2OS cells drugged overnight with 330 nM nocodazole in CO2 Independent Medium (Gibco). SiR-Hoechst DNA dye (Spirochrome) was added into the nocodazole containing media 2 h before imaging. Cells were released from nocodazole by washing three times with PBS and then placed in CO2 Independent Media (Gibco) containing SiR-Hoechst DNA dye (Spirochrome). Where indicated, 0.5 μM Reversine was added to cells after release. Cells were imaged every 2 min for 12 h in a heated chamber (37°C) with a Deltavision Elite System. Images were acquired as Z-sections (using the softWoRx software from Deltavision) and converted into maximal intensity projections TIFF files for illustrative purposes.
Pesenti M.E., Prumbaum D., Auckland P., Smith C.M., Faesen A.C., Petrovic A., Erent M., Maffini S., Pentakota S., Weir J.R., Lin Y.C., Raunser S., McAinsh A.D, & Musacchio A. (2018). Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80. Molecular Cell, 71(6), 923-939.e10.
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4

GFP-tagged Protein Microirradiation Imaging

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U2OS or HEK293 cells expressing GFP–FAM35A or GFP–C20ORF196 were cultured at 37 °C in CO2-independent medium (Invitrogen) containing 10% FBS in a temperature-controlled container in glass-bottom dishes (MatTek). Laser microirradiation was carried out with the Micro-Point Laser Illumination and Ablation System (ANDOR) coupled to a Leica DMI8 microscope with a 63 × CS2 oil immersion objective. Images were acquired with ANDOR IQ3 software through an ANDOR IXON camera with ANDOR Dragonfly system.
Gao S., Feng S., Ning S., Liu J., Zhao H., Xu Y., Shang J., Li K., Li Q., Guo R, & Xu D. (2018). An OB-fold complex controls the repair pathways for DNA double-strand breaks. Nature Communications, 9, 3925.
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5

Time-Lapse Migration Assay for Primary Fibroblasts

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Time-lapse video migration experiments were performed as described with modifications [28 (link)]. For migration experiments, primary human fibroblast cells were plated on 35 mm2 culture dishes with CO2-independent medium (Invitrogen) and incubated for 24 hours. Immediately following irradiation, time-lapse images of the cell migration were captured every 30 minutes for 4 hour in an environmental chamber monitored under microscopy (ECLIPSE TE2000-E, Nikon Instruments, Tokyo, Japan) with the temperature maintained at 37 °C. The time-lapse videos were generated using Volocity Image Software (PerkinElmer) and were analyzed using Openlab Software (PerkinElmer) to measure cell migration speed. “Migration speed” is the average speed in μm per minute that the cells travel in a 4-hour period. The migration speed of 50 cells was measured from each group. Statistical analysis of data was performed using the paired two-tailed Student s t-test, significance level was set at p < 0.05. Migration experiments were repeated three times to verify data reproducibility and accuracy.
Mamalis A., Garcha M, & Jagdeo J. (2015). Light Emitting Diode-Generated Blue Light Modulates Fibrosis Characteristics: Fibroblast Proliferation, Migration Speed, and Reactive Oxygen Species Generation. Lasers in surgery and medicine, 47(2), 210-215.
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6

Live-cell Imaging of Cell Migration

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Cells were plated in 12-well glass-bottomed imaging plates (MatTek) and cultured in CO2-independent medium (Invitrogen). Cell images were acquired using the Nikon TE2000-PFS inverted microscope (Nikon) enclosed in a humidified chamber maintained at 37°C. Cells were imaged every 10 minutes immediately after cell seeding with a 20× objective on five randomly selected fields per sample. Videos were generated with MetaMorph software (Molecular devices). For cell tracking analysis, the migration paths of 100 cells per sample were traced between 18 to 72 hours after cell seeding. Data were analyzed by Image-Pro Plus 6.0 (MediaCybernetics).
Wong C.M., Wei L., Au S.L., Fan D.N., Zhou Y., Tsang F.H., Law C.T., Lee J.M., He X., Shi J., Wong C.C, & Ng I.O. (2015). MiR-200b/200c/429 subfamily negatively regulates Rho/ROCK signaling pathway to suppress hepatocellular carcinoma metastasis. Oncotarget, 6(15), 13658-13670.
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7

Assay for Cellular Signaling Pathways

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Tissue culture dishes (35-mm) were purchased from Corning Life Sciences (Tewksbury, MA). The Dulbecco's Modified Eagle's Medium, CO2-independent medium, fetal bovine serum, phosphate buffered saline (PBS), and antibiotic-antimycotic mixture (penicillin, streptomycin, and amphotericin B) were from Invitrogen Life Technology (Invitrogen, Carlsbad, CA). The picric acid and sirius red dye were obtained from Sigma-Aldrich (St. Louis, MO). The PI3K/Akt inhibitor LY294002 and antibodies against total and phosphorylated forms of Akt (Ser473) and Erk1/2 (Thr202/Tyr204) were from Cell Signaling (Beverly, MA). Dihydrorhodamine (DHR) was from Calbiochem/Millipore (San Diego, CA).
Mamalis A., Koo E., Garcha M., Murphy W.J., Isseroff R.R, & Jagdeo J. (2016). High Fluence Light Emitting Diode-Generated Red Light Modulates Characteristics Associated with Skin Fibrosis. Journal of biophotonics, 9(11-12), 1167-1179.
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8

Tracking Newly Synthesized SNAP-gp135 Protein

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Pre-existing SNAP tagged gp135 protein was blocked using 0.32μM cell-permeable SNAP cell block (“BG block,” New England Biolabs) in serum-free medium at 37°C. Following removal of BG block by washing, cells were incubated for 30 min at 37°C to permit the synthesis of a new unblocked cohort of SNAP-gp135. Cells were washed with CO2-independent medium (Invitrogen) and suspended in a 19°C water bath for 80 min. During the last 20 min of this incubation, 150μg/ml cycloheximide was added to the medium to ensure that all newly synthesized protein had time to exit from the ER and that no new protein was synthesized. Cells were then washed twice with 37°C CO2-independent medium containing 150μg/ml cycloheximide and transferred to a 37°C water bath for 0 to 45 min.
In experiments using MyoVb-tail or Rab11-GFP, MDCK-S cells were transfected using Lipofectamine (Invitrogen) with the construct one day after being seeded onto Transwell filters.
Stoops E.H., Hull M, & Caplan M.J. (2016). Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments. Traffic (Copenhagen, Denmark), 17(12), 1272-1285.
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9

Laser-induced DNA Damage Assay

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Cells were irradiated with gamma rays from a cesium source (JL Shepherd and Associates). For laser irradiation, cells were pre-sensitized by overnight incubation with BrdU (10 μg/ml) and then micro-irradiated with a pulsed nitrogen laser (Spectra-Physics; 365nm, 10Hz) with output set at 70% of the maximum20 (link). Cells were maintained at 37°C in 35 mm glass-bottom culture dishes (MatTek Cultureware) during micro-irradiation and image acquisition; the growth medium was replaced by CO2-independent medium (Invitrogen) before irradiation.
Tomimatsu N., Mukherjee B., Hardebeck M.C., Ilcheva M., Camacho C.V., Harris J.L., Porteus M., Llorente B., Khanna K.K, & Burma S. (2014). Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nature communications, 5, 3561.
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10

Real-Time Recruitment of DNA Repair Factors

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GFP-NBS1, GFP-EXO1, GFP-Ku70, GFP-XLF, or GFP-XRCC4 were transfected into HCT116 DNA-PKcs +/+, −/−, or KD/− cells with JetPrime® (Polyplus) following the manufacturer's instructions. Twenty-four hours after the transfection laser micro-irradiation and real-time recruitment was performed with a Carl Zeiss Axiovert 200M microscope with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss) as previously described (28 (link)). DSBs were generated with a 365-nm pulsed nitrogen laser (Spectra-Physics), which was directly coupled to the epifluorescence path of the microscope (28 (link)). Time-lapse images were taken via a Carl Zeiss AxioCam HRm camera. The cells were maintained in a CO2-independent medium (Invitrogen) at 37°C during micro-irradiation and time-lapse imaging. Fluorescence intensities of the micro-irradiated area and control area were determined by Carl Zeiss Axiovision software, v4.5, and the intensity of irradiated was normalized to non-irradiated control area as previously described (26 (link)).
Lu H., Saha J., Beckmann P.J., Hendrickson E.A, & Davis A.J. (2019). DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response. Nucleic Acids Research, 47(18), 9467-9479.
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