Nahco3

Manufactured by Lonza

Sourced in Netherlands, Belgium

Sodium bicarbonate (NaHCO3) is a chemical compound commonly used in laboratory applications. It is a white, crystalline powder that serves as a versatile buffer and pH regulator. NaHCO3 is widely used in various scientific and analytical procedures to maintain specific pH levels and control chemical reactions.

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11 protocols using nahco3

Fibroblast Collagen Contraction Assay

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3000 fibroblasts were mixed with collagen matrix (1× EMEM [12-684, Lonza]; 10% FCS; 1× L-Glutamine; 1.0 mg/ml Collagen I; NaHCO₃ [17-613E, Lonza], diluted in PBS as required) and 300μl were plated in a 48 well plate and allowed to set. Images were taken at time 0 and used as control, and the collagen plug was separated from the well using a fine needle. After three days, wells were imaged again and contractility was measured based on the area of the collagen plug at day 3 normalized to area on day 0. Images were acquired on UVP Gel Doc-It Imager.

Kaur A., Ecker B.L., Douglass S.M., Kugel CH I.I.I., Webster M.R., Almeida F.V., Somasundaram R., Hayden J., Ban E., Ahmadzadeh H., Franco-Barraza J., Shah N., Mellis I.A., Keeney F., Kossenkov A., Tang H.Y., Yin X., Liu Q., Xu X., Fane M., Brafford P., Herlyn M., Speicher D.W., Wargo J.A., Tetzlaff M.T., Haydu L.E., Raj A., Shenoy V., Cukierman E, & Weeraratna A.T. (2018). Remodeling of the collagen matrix in aging skin promotes melanoma metastasis and affects immune cell motility. Cancer discovery, 9(1), 64-81.

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Isolation and Culture of HUVECs

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Human umbilical cords were freshly obtained from the KK Women's and Children's Hospital, Singapore, with the approval from the Singhealth Centralized Institutional Review Board (ref. no: CIRB Ref: 2014/323/D). The protocol to isolate and culture HUVECs was adapted from Baudin et al. (2007) (link). In brief, umbilical vein was flushed with 1× PBS to remove all red blood cells and digested with 2 mg/mL collagenase (Roche) solution for 10 min in 37°C saline bath. Cells were detached from the cord vein, plated onto LN521-coated plates, and cultured in M199 medium supplemented with 1% L-GlutaMAX, 1% penicillin-streptomycin, 15 mM HEPES (Gibco), 0.135% NaHCO₃ (Lonza), 30 μg/mL endothelial cell growth supplement (Sigma), 10 U/mL heparin (Merck), and 20% fetal bovine serum (Gibco). HUVECs were passaged onto new LN521-coated plates upon confluency. In this study, we compared the expression profiles of our hESC-derived cells with HUVECs at passage 1 to ensure the maintenance of their phenotypic features.

Nguyen M.T., Okina E., Chai X., Tan K.H., Hovatta O., Ghosh S, & Tryggvason K. (2016). Differentiation of Human Embryonic Stem Cells to Endothelial Progenitor Cells on Laminins in Defined and Xeno-free Systems. Stem Cell Reports, 7(4), 802-816.

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MDCK Cell Culture Conditions

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Madin-Darby canine kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM, Lonza Benelux BV, Breda, the Netherlands) supplemented with 10% fetal bovine serum (FBS) (Greiner), 100U/ml penicillin (P, Lonza), 100U/ml streptomycin (S, Lonza), 2mM L-glutamine (L-glu, Lonza), 1.5mg/ml sodium bicarbonate (NaHCO₃, Lonza), 10mM Hepes (Lonza) and 1X non-essential amino acids (NEAA, Lonza).

Richard M., Herfst S., van den Brand J.M., Lexmond P., Bestebroer T.M., Rimmelzwaan G.F., Koopmans M., Kuiken T, & Fouchier R.A. (2015). Low Virulence and Lack of Airborne Transmission of the Dutch Highly Pathogenic Avian Influenza Virus H5N8 in Ferrets. PLoS ONE, 10(6), e0129827.

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Cell Culture Conditions for Viral Studies

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Madin-Darby canine Kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM, Lonza Benelux BV, Breda, the Netherlands) supplemented with 10% foetal bovine serum (FBS) (Greiner), 100 U ml⁻¹ penicillin (PEN, Lonza), 100 U ml⁻¹ streptomycin (STR, Lonza), 2 mM L-glutamine (L-glu, Lonza), 1.5 mg ml⁻¹ sodium bicarbonate (NaHCO₃, Lonza), 10 mM Hepes (Lonza) and 1X non-essential amino acids (NEAA, Lonza). 293T cells (ATCC) were cultured in Dulbecco modified Eagle’s medium (DMEM, Lonza) supplemented with 10% FBS, 100 U ml⁻¹ PEN, 100 U ml⁻¹ SRT, 2mM L-glu, 1 mM sodium pyruvate (Gibco) and 1X NEAA. Human airway epithelia reconstituted in vitro (MucilAir^TM, EP02MP) were purchased from Epithelix Sàrl (Switzerland).

Richard M., van den Brand J.M., Bestebroer T.M., Lexmond P., de Meulder D., Fouchier R.A., Lowen A.C, & Herfst S. (2020). Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets. Nature Communications, 11, 766.

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MDCK Cell Culture Protocol

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MDCK cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM, Lonza Benelux BV, Breda, the Netherlands) supplemented with 10% fetal bovine serum (Greiner Bio-One B.V., Alphen aan den Rijn, The Netherlands.), 100 U/mL penicillin (P, Lonza), 100 U/mL streptomycin (S, Lonza), 2 mM L-glutamine (L-glu, Lonza), 1.5 mg/mL sodium bicarbonate (NaHCO₃, Lonza), 10 mM HEPES (Lonza), and 1× non-essential amino acids (NEAA, Lonza).

Herfst S., Begeman L., Spronken M.I., Poen M.J., Eggink D., de Meulder D., Lexmond P., Bestebroer T.M., Koopmans M.P., Kuiken T., Richard M, & Fouchier R.A. (2023). A Dutch highly pathogenic H5N6 avian influenza virus showed remarkable tropism for extra-respiratory organs and caused severe disease but was not transmissible via air in the ferret model. mSphere, 8(4), e00200-23.

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Quantifying SARS-CoV-2 Viral Titer via Plaque Assay

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VeroE6 cells were grown to a confluent monolayer in 6-well plates. A 10-fold dilution series of viral solutions was prepared in PBS (Sigma-Aldrich) containing 1% (v/v) penicillin/streptomycin, 0.6% (v/v) BSA (35%) (Sigma-Aldrich), 0.01% (w/v) CaCl₂ (1%), and 0.01% (w/v) MgCl₂ (1%) from the medium in which the organoids were incubated. The VeroE6 cells were incubated with the dilution series for 1 h at 37 °C. Afterward, the inoculum was replaced by plaque medium: 63% (v/v) 2× MEM 20% (v/v), 10× MEM (Gibco), 3.2% (v/v) NaHCO₃ (Lonza), 2% (v/v) HEPES (1 M; pH 7.2) (Sigma-Aldrich), 1.2% (v/v) BSA (35%), 1% (v/v) 100× penicillin/streptomycin/l-glutamine solution (10,000 U/mL penicillin; 10,000 μg/mL streptomycin, 29.2 mg/mL l-glutamine) (Gibco), 2% (v/v) FBS, and 35% (v/v) Agar (2%) (Oxoid). After 24–96 h, the plaques were counted.

Menuchin-Lasowski Y., Schreiber A., Lecanda A., Mecate-Zambrano A., Brunotte L., Psathaki O.E., Ludwig S., Rauen T, & Schöler H.R. (2022). SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids. Stem Cell Reports, 17(4), 789-803.

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Antifungal Susceptibility Testing for Candida parapsilosis

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MIC values were determined according to the M27-A3 protocol (41 ), and interpretation of MIC values was defined by the M27-S4 supplementary document (42 ). MICs were measured in RPMI 1640 with MOPS with L-Gln and without NaHCO₃ (catalog no. 04-525F; Lonza) after 24 h and 48 h. MIC values for echinocandins were defined as the lowest concentrations that resulted in at least 50% growth reduction. Azoles (fluconazole [FLU], voriconazole [VOR], posaconazole [POS], and itraconazole [ITR]; Sigma-Aldrich) and echinocandins (CAS from Sigma-Aldrich; AND and MICA from MedChem Express) were used to test the susceptibility of C. parapsilosis strains.

Papp C., Kocsis K., Tóth R., Bodai L., Willis J.R., Ksiezopolska E., Lozoya-Pérez N.E., Vágvölgyi C., Mora Montes H., Gabaldón T., Nosanchuk J.D, & Gácser A. (2018). Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance. mSphere, 3(6), e00547-18.

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Mosquito Homogenization and Preparation

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Mosquito pools were homogenized in 1 ml cold medium (DMEM with 4.5 g/l glucose (Lonza, Verviers, Belgium), NaHCO3 (0.075%, Lonza, Verviers, Belgium), Hepes buffered saline (17 mM, Lonza, Verviers, Belgium), penicillin/streptomycin (1000 U/ml, Lonza, Verviers, Belgium), amphotericin (0.0125 mg/ml, in house made)) with one 1/4″ ceramic sphere (MP Biomedicals, Solon OH, USA) using the FastPrep-24 5G Homogenizer MP Biomedicals (30″ 5 m/s). Mosquito pools were placed at +4 °C for at least 5 min before spinning down the samples for 1 min at 13,000 g.

Blom R., Schrama M.J., Spitzen J., Weller B.F., van der Linden A., Sikkema R.S., Koopmans M.P, & Koenraadt C.J. (2022). Arbovirus persistence in North-Western Europe: Are mosquitoes the only overwintering pathway?. One Health, 16, 100467.

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Cell Culture Preparation for Research

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Madin-Darby canine kidney (MDCK) cells (ATCC) were cultured in Eagle’s minimal essential medium (EMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Greiner), 100 U/ml penicillin (PEN) (Lonza), 100 U/ml streptomycin (STR) (Lonza), 2 mM l-glutamine (l-Glu) (Lonza), 1.5 mg/ml sodium bicarbonate (NaHCO₃) (Lonza), 10 mM HEPES (Lonza) and 1× nonessential amino acids (NEAA) (Lonza). 293T cells were cultured in Dulbecco modified Eagle’s medium (DMEM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 U/ml STR, 2 mM l-Glu, 1 mM NaHCO₃, and 1× NEAA. Vero cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza) supplemented with 10% FBS, 100 U/ml PEN, 100 mg/ml STR, and 2 mM l-Glu.

Herfst S., Mok C.K., van den Brand J.M., van der Vliet S., Rosu M.E., Spronken M.I., Yang Z., de Meulder D., Lexmond P., Bestebroer T.M., Peiris J.S., Fouchier R.A, & Richard M. (2018). Human Clade 2.3.4.4 A/H5N6 Influenza Virus Lacks Mammalian Adaptation Markers and Does Not Transmit via the Airborne Route between Ferrets. mSphere, 3(1), e00405-17.

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SARS-CoV-2 Propagation and Characterization

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SARS-CoV-2 (isolate BetaCoV/Munich/BavPat1/2020; GISAID ID EPI_ISL 406862; kindly provided by Prof. Dr. C. Drosten) was propagated to passage 3 on VeroE6 cells (ATCC) in Opti-MEM I (1×) + GlutaMAX (Gibco), supplemented with penicillin (10,000 IU mL⁻¹, Lonza) and streptomycin (10,000 IU mL⁻¹, Lonza) at 37 °C in a humidified CO2 incubator. VeroE6 cells were inoculated at an moi of 0.01. Supernatant was harvested 72 hpi, cleared by centrifugation and stored at –80 °C. The virus stock was tested mycoplasma negative and contained 3.15 × 10⁸ genome copies/ml (RdRp gene).
VeroE6 cells were maintained in Dulbecco modified Eagle medium (DMEM, Gibco) supplemented with 10% foetal calf serum (Greiner), 2 mM of L-glutamine (Gibco), 10 mM Hepes (Lonza), 1.5 mg ml⁻¹ sodium bicarbonate (NaHCO₃, Lonza), penicillin (10,000 IU/mL) and streptomycin (10,000 IU/mL) at 37 °C in a humidified CO₂ incubator. All work was performed in a Class II Biosafety Cabinet under BSL-3 conditions at the Erasmus Medical Center.

Richard M., Kok A., de Meulder D., Bestebroer T.M., Lamers M.M., Okba N.M., Fentener van Vlissingen M., Rockx B., Haagmans B.L., Koopmans M.P., Fouchier R.A, & Herfst S. (2020). SARS-CoV-2 is transmitted via contact and via the air between ferrets. Nature Communications, 11, 3496.

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