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Agilent seahorse xf96 cell culture microplate

Manufactured by Agilent Technologies
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The Agilent Seahorse XF96 Cell Culture Microplate is a laboratory equipment designed for cell culture applications. It provides a standardized platform for measuring cellular metabolic activity.

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Lab products found in correlation

Carnitine hydrochloride, by Merck Group (1 mentions) Rs 2000 small animal irradiator, by Rad Source (1 mentions) Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions) Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions) Wave software, by Agilent Technologies (1 mentions)

4 protocols using agilent seahorse xf96 cell culture microplate

1

Seahorse Analysis of NIPSNAP1/2 Knockdown

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The day before siRNA transfection, BEAS-2B cells were seeded into a 12-well plate at a density of 1.0 × 105 cells per well. After incubation overnight, cells were transfected with siRNAs against NIPSNAP1 and 2 using RNAiMAX. The next day, 2.0 × 104 cells were re-seeded into a homemade collagen-coated Agilent Seahorse XF96 Cell Culture Microplate (101085-004; Agilent Technologies, Santa Clara, CA, USA) and then incubated overnight. To measure the OCR and ECAR after CAM treatment, 2.0 × 104 BEAS-2B cells were seeded into a homemade collagen-coated Agilent Seahorse XF96 Cell Culture Microplate. After incubation for 8 h, CAM was added to each well and cells were further incubated overnight. The mitochondrial OCR and ECAR were measured using Extracellular Flux Assay Kits and a Seahorse XF Cell Mito Stress Test kit (103015-100, Agilent Technologies) with the Seahorse XFe96 analyzer (Agilent Technologies) according to the manufacturer's instructions. During the measurement, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a mixture of rotenone and antimycin A were sequentially injected at final concentrations of 1, 1, and 2 μM, respectively. The bioenergetic profiles were calculated using Wave software (Ver. 2.6.3; Agilent Technologies).
Yamamoto S., Ogasawara N., Mitsuhashi Y., Takano K, & Yokota S.I. (2024). The clarithromycin-binding proteins NIPSNAP1 and 2 regulate cytokine production through mitochondrial quality control. Scientific Reports, 14, 2354.
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2

Fatty Acid Oxidation Assay in BMDMs

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The ability of the cells to oxidize exogenous fatty acid substrates was measured using the Agilent Seahorse XF Cell Mito Stress Test kit according to manufacturer’s recommendations and the data analyzed using the Agilent Seahorse XF Wave software tool as described in the previous section. BMDMs (4x105 cells) were seeded in Agilent Seahorse XF96 cell culture microplate and allowed to adhere in complete culture medium. The growth medium was then replaced and incubated for 24 hours with substrate limited DMEM medium containing reduced concentrations of glucose (0.5mM), glutamax (1mM) and fetal bovine serum (1%) to deplete endogenous substrates within the cells. Carnitine hydrochloride (0.5mM) (Sigma, St. Louis, MO) was added fresh during the media change. The media was then replaced with FAO assay medium containing 111mM NaCl, 4.7mM KCl, 1.25mM CaCl2, 2mM MgSO4, 1.2mM NaH2PO4 supplemented with 2.5mM glucose, 0.5mM Carnitine and 5mM HEPES and incubated for 30-45 minutes. XF Palmitate BSA or BSA control was added to the medium just prior to the start of the XF assay. The OCR (pmol of O2/min) was measured after sequential addition of electron transport chain modulators Oligomycin, FCCP and Rotenone/Antimycin A.
Wessendarp M., Watanabe-Chailland M., Liu S., Stankiewicz T., Ma Y., Kasam R.K., Shima K., Chalk C., Carey B., Rosendale L.R., Filippi M.D, & Arumugam P. (2021). Role of GM-CSF in regulating metabolism and mitochondrial functions critical to macrophage proliferation. Mitochondrion, 62, 85-101.
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3

Irradiation Impact on Salivary Gland ATP

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Primary cells isolated from parotid salivary glands were plated on an Agilent Seahorse XF96 Cell Culture Microplate (Part No. 101085-004, Agilent Technologies, Cedar Creek, TX) at a seeding density of 150,000 cells/well and cultured prior to irradiation at 24 h or 48 h before running the Seahorse ATP Rate Assay. Cells were exposed to a single dose of 5 Gy irradiation (X-ray, RS 2000 Small Animal Irradiator, Rad Source). The untreated cells were shielded with > 6 mm lead.
Buss L.G., Rheinheimer B.A, & Limesand K.H. (2024). Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands. Scientific Reports, 14, 845.
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4

Mitochondrial Respiration Profiling in Cells

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A total of 40,000 cells were seeded per well on Agilent Seahorse XF96 cell culture microplate (Agilent USA 101085-004). Sensor Cartridge was hydrated the overnight before measuring mitochondrial cellular respiration. Cells were recovered in Agilent Seahorse XF Base medium (pH 7.4) (Agilent USA 103334-100) supplemented with 20 mM glucose (Sigma USA G8270) and 2 mM pyruvate (Sigma USA S8636). Oxygen consumption rates were measured by CF Analyzer (Searhorse Bioscience) at 37 °C with the injection of 2 µM oligomycin, 0.5 µM FCCP or 7.5 µM ND-Nic, and 0.5 µM antimycin A into the ports of the XF assay cartridge (Agilent 103729-100).
Ngai T.W., Elfar G.A., Yeo P., Phua N., Hor J.H., Chen S., Ho Y.S, & Cheok C.F. (2021). Nitro-Deficient Niclosamide Confers Reduced Genotoxicity and Retains Mitochondrial Uncoupling Activity for Cancer Therapy. International Journal of Molecular Sciences, 22(19), 10420.
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