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Cell culture microplate

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The cell culture microplate is a multi-well plate designed for growing and maintaining cell cultures. It provides a standardized platform for culturing cells in a controlled and organized manner. The microplate consists of multiple individual wells, allowing for parallel experimentation and observation of cell growth and behavior.

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Lab products found in correlation

Oligomycin, by Merck Group (2 mentions) Seahorse xf96 flux analyzer, by Agilent Technologies (2 mentions) Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (2 mentions) Laminin 1, by Bio-Techne (2 mentions) Xf assay medium, by Agilent Technologies (2 mentions) Antimycin a, by Merck Group (2 mentions) Rotenone, by Merck Group (2 mentions) Xfe24 extracellular flux analyzer, by Agilent Technologies (2 mentions) Seahorse xf96 extracellular flux analyzer, by Agilent Technologies (2 mentions) Cell tak, by Corning (2 mentions) Seahorse xf assay medium, by Agilent Technologies (2 mentions) Seahorse xf glycolytic stress assay kit, by Agilent Technologies (2 mentions) Xf cell mito stress test kit, by Agilent Technologies (2 mentions) Glutamine, by Agilent Technologies (1 mentions) Base medium, by Agilent Technologies (1 mentions) Glucose, by Agilent Technologies (1 mentions) Sodium pyruvate, by Agilent Technologies (1 mentions) Seahorse xfe24, by Agilent Technologies (1 mentions) Seahorse medium, by Agilent Technologies (1 mentions) Oligomycin, by Agilent Technologies (1 mentions)

12 protocols using cell culture microplate

1

Seahorse Bioenergetic Analysis of COPD Airway Cells

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Extracellular acidification rates and oxygen consumption rates were determined by the Seahorse XF 96 flux analyzer (Seahorse Bioscience). Primary airway epithelial cells were isolated as described above and plated onto cell culture microplates (Seahorse Bioscience) coated with 50 ng/μl Laminin 1 (3400-010-01, Trevigen) for 4 days (media change on Day 2). On the day of the experiment cells were treated with CSE for 4 hours. Cells were incubated in XF assay medium (Seahorse bioscience), supplemented with 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for one hour prior to the measurement. After the recording of the basal rates of ECAR and OCR, final concentrations of 1 μM oligomycin, 2 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and 0.5–0.5 μM rotenone and antimycin A were added (Sigma) through the instruments injection ports in order to obtain proton leak, maximal respiratory capacity and non-mitochondrial respiration respectively. Rates were normalized by DNA with Hoechst 33342 against standards with known concentrations of DNA.
Cloonan S.M., Glass K., Laucho-Contreras M.E., Bhashyam A.R., Cervo M., Pabón M.A., Konrad C., Polverino F., Siempos I.I., Perez E., Mizumura K., Ghosh M.C., Parameswaran H., Williams N.C., Rooney K.T., Chen Z.H., Goldklang M.P., Yuan G.C., Moore S.C., Demeo D.L., Rouault T.A., D’Armiento J.M., Schon E.A., Manfredi G., Quackenbush J., Mahmood A., Silverman E.K., Owen C.A, & Choi A.M. (2016). Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice. Nature medicine, 22(2), 163-174.
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2

Seahorse Bioenergetic Analysis of COPD Airway Cells

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Extracellular acidification rates and oxygen consumption rates were determined by the Seahorse XF 96 flux analyzer (Seahorse Bioscience). Primary airway epithelial cells were isolated as described above and plated onto cell culture microplates (Seahorse Bioscience) coated with 50 ng/μl Laminin 1 (3400-010-01, Trevigen) for 4 days (media change on Day 2). On the day of the experiment cells were treated with CSE for 4 hours. Cells were incubated in XF assay medium (Seahorse bioscience), supplemented with 5 mM glucose, 4 mM glutamine and 1 mM pyruvate for one hour prior to the measurement. After the recording of the basal rates of ECAR and OCR, final concentrations of 1 μM oligomycin, 2 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and 0.5–0.5 μM rotenone and antimycin A were added (Sigma) through the instruments injection ports in order to obtain proton leak, maximal respiratory capacity and non-mitochondrial respiration respectively. Rates were normalized by DNA with Hoechst 33342 against standards with known concentrations of DNA.
Cloonan S.M., Glass K., Laucho-Contreras M.E., Bhashyam A.R., Cervo M., Pabón M.A., Konrad C., Polverino F., Siempos I.I., Perez E., Mizumura K., Ghosh M.C., Parameswaran H., Williams N.C., Rooney K.T., Chen Z.H., Goldklang M.P., Yuan G.C., Moore S.C., Demeo D.L., Rouault T.A., D’Armiento J.M., Schon E.A., Manfredi G., Quackenbush J., Mahmood A., Silverman E.K., Owen C.A, & Choi A.M. (2016). Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice. Nature medicine, 22(2), 163-174.
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3

Cellular Respiration Quantification via OCR

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Cellular oxygen consumption rates (OCR) were determined in an XFe 24 Extracellular Flux Analyzer (Agilent Seahorse Bioscience). After transfection, cells were trypsinized and seeded at 60,000 cells per well of cell culture microplates (Seahorse Bioscience) and incubated at 37 °C and 5% CO2 overnight. The XF Base Medium (103334-100) with substrates of 25 mM glucose, 0.5 mM pyruvate, and 2.5 mM L-glutamine was freshly prepared and pH adjusted to 7.4. OCR was measured after 2 μg/ml oligomycin (Oligo), 1 μM carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), 4 μM Antimycin A (AA) and 1 μM rotenone (Rot) sequential injection. After the measurement, OCR was normalized to protein content.
Zhang K., Bao R., Huang F., Yang K., Ding Y., Lauterboeck L., Yoshida M., Long Q, & Yang Q. (2021). ATP synthase inhibitory factor subunit 1 regulates islet β-cell function via repression of mitochondrial homeostasis. Laboratory investigation; a journal of technical methods and pathology, 102(1), 69-79.
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4

Cellular Respiration Quantification via OCR

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Cellular oxygen consumption rates (OCR) were determined in an XFe 24 Extracellular Flux Analyzer (Agilent Seahorse Bioscience). After transfection, cells were trypsinized and seeded at 60,000 cells per well of cell culture microplates (Seahorse Bioscience) and incubated at 37 °C and 5% CO2 overnight. The XF Base Medium (103334-100) with substrates of 25 mM glucose, 0.5 mM pyruvate, and 2.5 mM L-glutamine was freshly prepared and pH adjusted to 7.4. OCR was measured after 2 μg/ml oligomycin (Oligo), 1 μM carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), 4 μM Antimycin A (AA) and 1 μM rotenone (Rot) sequential injection. After the measurement, OCR was normalized to protein content.
Zhang K., Bao R., Huang F., Yang K., Ding Y., Lauterboeck L., Yoshida M., Long Q, & Yang Q. (2021). ATP synthase inhibitory factor subunit 1 regulates islet β-cell function via repression of mitochondrial homeostasis. Laboratory investigation; a journal of technical methods and pathology, 102(1), 69-79.
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5

Mitochondrial Respiration Profiling in Cells

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OCR was assessed using the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). Cells were seeded quadruplicately in cell culture microplates (Agilent Technologies, Santa Clara, CA, USA) and treated with control or HP for 24 h. One hour prior to the assay, the culture medium was replaced by base medium (Agilent Technologies) supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and 10 mM glucose (Agilent Technologies), and then cells were incubated at 37 °C without CO2. After three basal respiration measurements without additives, the ATP synthase inhibitor (1 μM oligomycin) was added, followed by subsequent mitochondrial uncoupler (1 μM FCCP), and complex I and III inhibitors (1 μM rotenone and 1 μM antimycin A), respectively. Three separate measurements were recorded after the reagents (all bought from Agilent Technologies) were injected.
Huang Y., Wang S., Zhou J., Liu Y., Du C., Yang K., Bi X., Liu M., Han W., Wang K., Xiong J., Wang S., Wang Y., Nie L., Liu C., Zhang D., Gu J., Zeng C, & Zhao J. (2020). IRF1-mediated downregulation of PGC1α contributes to cardiorenal syndrome type 4. Nature Communications, 11, 4664.
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6

Quantifying Cellular Respiration and Glycolysis

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After a preliminary experiment determining the proper cell density, the cells were seeded on Cell Culture Microplates (Agilent) and Calibrate Sensor Cartridges were treated with Calibrant for 10 h, followed by twice washing using OCR buffer solution or ECAR buffer solution, respectively. Next, the culture plate was placed under the Calibrate Sensor Cartridges and OCR detection reagent (2 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone) or ECR detection reagent (10 mM glucose, 1 μM oligomycin, 50 mM 2-DG) was added in Calibrate Sensor Cartridges. The values were measured in Seahorse XFe24 (Agilent). At last, the cells were lysed with RIPA for BCA quantification to normalize the total cell numbers.
Qi Y., Ye Y., Wang R., Yu S., Zhang Y., Lv J., Jin W., Xia S., Jiang W., Li Y, & Zhang D. (2022). Mitochondrial dysfunction by TFAM depletion disrupts self-renewal and lineage differentiation of human PSCs by affecting cell proliferation and YAP response. Redox Biology, 50, 102248.
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7

Measuring Glycolytic Capacity in CAR-T Cells

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CAR‐T cells were resuspended in Seahorse XF Assay Medium (Agilent Technologies) and seeded at 6000 cells per well in a 96‐well plate. The cell culture microplate (101085‐004, Agilent) was pre‐coated with Cell‐Tak (354240, Corning) for 20 min at room temperature. The XF 96 sensor cartridge was hydrated with 200 µL of calibration buffer per well in a 37 °C non‐CO2 incubator overnight. The extracellular acidification rate (ECAR) was measured in real time in an XFe96 analyzer using a Seahorse XF Glycolytic Stress Assay Kit (103020‐100, Agilent). The rate of glycolysis under basal conditions was measured following the first addition of glucose (10 mm). The cellular maximum glycolytic capacity was defined as the subsequent increase in ECAR after oligomycin (1.5 mm) injection and glycolytic reserve was determined by subtracting the rate of glycolysis before and after addition of 2‐deoxy‐D‐glucose (50 mm).
Shao M., Teng X., Guo X., Zhang H., Huang Y., Cui J., Si X., Ding L., Wang X., Li X., Shi J., Zhang M., Kong D., Gu T., Hu Y., Qian P, & Huang H. (2022). Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti‐Leukemia Efficacy of CAR‐T Cells via SOCE‐Calcineurin‐NFAT and Glycolysis Pathways. Advanced Science, 9(9), 2103508.
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8

Mitochondrial Stress Assay Protocol

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80,000 cells per well were seeded in Agilent cell culture microplate and incubated in 37°C at 5% CO2 overnight. Probe sensitization, assay buffer preparation, and program settings were followed as suggested by Seahorse XF Cell Mito Stress Test Kit’s (catalogue number 103015–100) user guide. After the cells adhered, cells were treated with compound for 30 min. Oligomycin, FCCP, and Rotenone/ Antimycin A was added at a final concentration of 2.5 µM, 2 µM, and 0.5 µM, respectively.
Gowans F.A., Thach D.Q., Wang Y., Altamirano Poblano B.E., Dovala D., Tallarico J.A., McKenna J.M., Schirle M., Maimone T.J, & Nomura D.K. (2023). Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells. bioRxiv.
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9

Seahorse XF Cell Mito Stress Test

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40,000 cells per well were seeded in Agilent cell culture microplate and incubated in 37°C at 5% CO2 overnight. Probe sensitization, assay buffer preparation, and “induced” program settings were followed as suggested by Seahorse XF Cell Mito Stress Test Kit’s user guide (catalogue no. 103592–100). Oligomycin and Rotenone/Antimycin A was added at a final concentration of 1.5 µM and 0.5 µM, respectively.
Gowans F.A., Thach D.Q., Wang Y., Altamirano Poblano B.E., Dovala D., Tallarico J.A., McKenna J.M., Schirle M., Maimone T.J, & Nomura D.K. (2023). Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells. bioRxiv.
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10

Synaptosomal Respiratory Monitoring

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For monitoring respiration, synaptosomes were resuspended in Seahorse medium (Agilent Technologies) (phenol-free DMEM pH 7.4, supplemented with 2 mM glutamine, 10 mM glucose, 1 mM pyruvate). Eighty μg synaptosomal protein per well was aliquoted into a 24 well of a cell culture microplate (Agilent Technologies) coated with a 1:15,000 diluted polyethyleneimine (PEI). The plate was centrifuged at 3,000g for 1 h at 4°C. The cell culture microplate was incubated and loaded into the Seahorse XF24 extracellular flux analyzer following the manufacturer’s nstructions. All experiments were performed at 37°C. Reagents were added at appropriate dilutions in Seahorse medium (volumes and concentrations provided in Supplemental table 2). Three biological replicates and four technical replicates were run per plate.
Cheslow L., Byrne M., Kopenhaver J.S., Iacovitti L., Smeyne R.J., Snook A.E, & Waldman S.A. (2023). GUCY2C signaling limits dopaminergic neuron vulnerability to toxic insults. Research Square.
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