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Synergy neo model neoalhpa b

Manufactured by Agilent Technologies
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The Synergy Neo model NEOALHPA B is a laboratory equipment product manufactured by Agilent Technologies. It serves as a multi-mode microplate reader, capable of performing various spectroscopic and luminescent measurements.

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Lab products found in correlation

Oligomycin a, by Bio-Techne (2 mentions) Atp fluorometric assay kit, by Abcam (2 mentions) 10 kd spin column, by Thermo Fisher Scientific (2 mentions) Compound c, by Merck Group (2 mentions) 2 nbdg, by Cayman Chemical (2 mentions)

4 protocols using synergy neo model neoalhpa b

1

Intracellular ATP Modulation by E-cadherin

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Cells were plated at a density of 0.75 ×105 for 2 days in 35mm dishes. An hour before applying force, cells were treated with Compound C (Sigma 10μM), Oligomycin A (Tocris, 10μM), 2-NBDG (fluorescently-labeled 2-deoxyglucose from Cayman Chemical, 150μg/mL). Dynabeads (0.15mg) coated with 1μg of Fc-E-cadherin or IgG were incubated with the cells for 45 minutes. Force was applied using a ceramic magnet for 10 minutes. Intracellular ATP levels were examined using a Fluorometric ATP assay kit from Abcam (ab83355). Cells were lysed in 200μL of ATP Assay Buffer, centrifuged at 12,000 rpm for 5 minutes at 4°C, and protein was removed from the supernatant using a 10 Kd spin column (Thermo Scientific). 5μL of the de-proteinated sample were added to ATP reaction mix in 96-well plates and a fluorescence reading at 535/587nm was made (Biotek Synergy Neo model NEOALHPA B, Gen 5 software).
Bays J.L., Campbell H.K., Heidema C., Sebbagh M, & DeMali K.A. (2017). Linking E-cadherin mechanotransduction to cell metabolism through force mediated activation of AMPK. Nature cell biology, 19(6), 724-731.
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2

Quantifying Glucose Uptake under Shear Stress

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Glucose uptake was assessed by measuring the fluorescence of 2-NBDG (Cayman Chemicals 11046). Cells were grown to confluency. Cells were pretreated with inhibitors or function blocking antibodies for 1 hour in glucose free media with 0.1mg/ml of 2-NBDG. Cells were then left under no shear or shear conditions for an additional hour. After incubation time the cells were washed 3x with PBS and lysed in EB lysis buffer (1 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 20 μg/mL aprotinin, 2 mM Na3VO4, and 1 mM PMSF). Lysate was clarified by spinning down for 10 minutes at 12,000 rcf. Supernatant was transferred to 96 well plate and a fluorescence reading of 485/535 was taken (Biotek Synergy Neo model NEOALHPA B, Gen 5 software).
Cronin N.M., Dawson L.W, & DeMali K.A. (2024). Mechanical activation of VE-cadherin stimulates AMPK to increase endothelial cell metabolism and vasodilation. bioRxiv.
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3

Intracellular ATP Modulation by E-cadherin

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Cells were plated at a density of 0.75 ×105 for 2 days in 35mm dishes. An hour before applying force, cells were treated with Compound C (Sigma 10μM), Oligomycin A (Tocris, 10μM), 2-NBDG (fluorescently-labeled 2-deoxyglucose from Cayman Chemical, 150μg/mL). Dynabeads (0.15mg) coated with 1μg of Fc-E-cadherin or IgG were incubated with the cells for 45 minutes. Force was applied using a ceramic magnet for 10 minutes. Intracellular ATP levels were examined using a Fluorometric ATP assay kit from Abcam (ab83355). Cells were lysed in 200μL of ATP Assay Buffer, centrifuged at 12,000 rpm for 5 minutes at 4°C, and protein was removed from the supernatant using a 10 Kd spin column (Thermo Scientific). 5μL of the de-proteinated sample were added to ATP reaction mix in 96-well plates and a fluorescence reading at 535/587nm was made (Biotek Synergy Neo model NEOALHPA B, Gen 5 software).
Bays J.L., Campbell H.K., Heidema C., Sebbagh M, & DeMali K.A. (2017). Linking E-cadherin mechanotransduction to cell metabolism through force mediated activation of AMPK. Nature cell biology, 19(6), 724-731.
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4

Quantifying Cellular Glucose Uptake

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Glucose uptake was measured using a kit from Cayman Chemicals (600470) and used as previously described11 (link). For all lysates, 100 μL were loaded in triplicate onto a 96-well plate and a fluorescence reading of 485/535 was taken (Biotek Synergy Neo model NEOALHPA B, Gen 5 software). The concentration of glucose was determined using a standard curve and results are reported as micrograms per milliliter per 105 cells.
Salvi A.M., Bays J.L., Mackin S.R., Mege R.M, & DeMali K.A. (2021). Ankyrin G Organizes Membrane Components to Promote Coupling of Cell Mechanics and Glucose Uptake. Nature cell biology, 23(5), 457-466.
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