The drug release behavior of CP-DDA NPs was studied under four different conditions: pH7.4, pH7.4 with 2 uM GSH, pH6.0 and pH 6.0 with 10 mM GSH. Briefly, 0.5 ml of CP-DDA NPs (800 ug) was dialyzed with 10 ml PBS (pH 7.4 or the other conditions mentioned above) in a dialysis bag (MWCO 1000 Da) at 37 ± 0.2 °C. PBS contains 10% ethanol to promote the dissolution of DAS. Dialysate (10 ml) was collected and replenished with 10 ml of fresh buffer at predetermined time. The amounts of DAS released from CP-DDA NPs were measured using SpectraMax M2/M2e Microplate Readers (Molecular Devices, USA) at 320 nm. The amounts of CP released from CP-DDA NPs were measured using high performance liquid chromatography (HPLC, Thermo Scientific UltiMate 3000) with a diode-array detector and a C18 column (15 cm × 0.46 cm, Syncronis). 1 ml the release solution was taken, 200 ul of DDTC solution (5%) was added, vortex for 1 min, and then incubate in a 37 °C water bath for 30 min. 1 ml of chloroform was added for extraction, vortexed and mixed for 1 min, and centrifuged at 10 000 rpm for 10 min. Next, the lower layer of chloroform was aspirated and filtered. Detection wavelength was 254 nm; mobile phase: methanol: water (75:25, v/v); flow rate 1 ml/min; column temperature: 25 °C; injection volume: 10 ul.
Spectramax m2 m2e microplate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M2/M2e Microplate Reader is a high-performance multi-mode microplate reader designed for a variety of absorbance, fluorescence, and luminescence-based assays. It offers flexible detection capabilities and a compact, benchtop footprint.
Automatically generated - may contain errors
Lab products found in correlation
Las 3000 imaging system, by Fujifilm (2 mentions)
Ettan dige imager, by GE Healthcare (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate bcip, by Merck Group (1 mentions)
Alkaline phosphatase assay kit, by Abcam (1 mentions)
Nitroblue tetrazolium nbt, by Merck Group (1 mentions)
Centrifuge 5417r, by Eppendorf (1 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Protein lobind tube, by Eppendorf (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Cell counting kit 8 wst 8 cck8 reagent, by GLPBIO (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by AppliChem (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Formaldehyde solution, by Merck Group (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Amplex red, by Molecular Devices (1 mentions)
H2dcfda, by Molecular Devices (1 mentions)
18 protocols using spectramax m2 m2e microplate reader
1
Drug Release Behavior of Nanoparticles
2
BHK-21 Cell Viability Assay
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BHK-21 cells were seeded in 96-well plates at 2 × 104 cells per well, and then, the cells were incubated in RPMI 1640 medium containing 2% FBS in the presence of Cur and Cur-CQDs (0 − 1000 μg ml−1) for 24 h. The medium was replaced with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo; 50 μl;) and incubated at 37 °C for 5 h. DMSO (Merck KGaA; 150 μl) was then added and shaken at 37 °C for 5 min. The absorbance at 570 nm was detected by SpectraMax M2/M2e Microplate Readers (Molecular Device) for cell counting.
3
Cell Viability Assay of Artemisia annua
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For the cell viability test, RAW264.7 cells were seeded into 96‐well plates at a density of 1 × 105 cells/ml and cultured in DMEM for 24 hr. After cells were attached, various concentrations of A. annua water extract (AAWE) was applied to each well and cultured for another 24 hr. Medium was replaced with fresh medium containing 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT) (AppliChem), and plates were incubated for 4 hr in a dark condition at 37°C. After medium was removed, insoluble formazan was dissolved with 100 μl of dimethyl sulfoxide and shaken for 30 min. The optical absorbance of the dissolved sample was measured at 540 nm with an ELISA plate reader (SpectraMax M2/M2e Microplate Readers; Molecular Devices).
4
Quantifying IDO Enzyme Activity
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IDO is the enzyme responsible for the transformation of tryptophan into kynurenine. It is possible to determine the biological activity of the enzyme by measuring kynurenine levels in the cell supernatant [44] or in cell lysates [45] . The cells were lysed through freeze-thaw (5 • ) cycles in 100 mL of culture medium. Subsequently, the cells were centrifuged at 2,000 rpm, for 5 min, and 80 mL of the supernatant were transferred to a new tube. Eighty microliters of 2 • IDO buffer (100 mM PBS, 40 mM ascorbate, 20 mM methylene blue, 200 mg/mL catalase, 800 mM L-Tryptophan, pH 6.5) was added to the supernatant, and it was incubated for 30 min at 37°C. The supernatant was mixed with 50 mL of 30% trichloroacetic acid to stop the reaction, and a further incubation of 30 min at 52°C was performed. After centrifugation at 8,000 g for 5 min, the supernatant (or standard curve) was mixed with an equal amount of Ehrlich's reagent (100 mg of p-dimethylbenzaldehyde in 5 mL of glacial acetic acid) and added to a straight-bottom 96-well plate. The absorbance reading was performed at a wavelength of 492 nm on a plate reader (SpectraMax M2/M2e Microplate Readers; Molecular Devices). Serial dilutions of L-kynurenine were used as standards (0, 6.25, 12.5, 25, 50, 100, 200, and 400 mM).
5
Measuring Cell Death via LDH Assay
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Lactate dehydrogenase (LDH) contents were measured in supernatants of control or treated SCAP, as a biochemical marker of cell death [43] . The cells were treated with LPS, poly(i:c), or IFN-g, as described earlier. The LDH activity was measured by using a commercially available kit, according to the manufacturer's recommendations (Labtest, Lagoa Santa, Minas Gerais, Brazil). The absorbance was read at 340 nm in a spectrophotometer (SpectraMax M2/ M2e Microplate Readers; Molecular Devices, San Jose, CA). The protein content was determined by the biuret method (total protein monoreagent kit, Bioclin), and the values of LDH were normalized by the protein content of the same sample.
6
Evaluating Osteogenic Differentiation via ALP Activity
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At 7–9 days after osteogenic differentiation, the medium was removed, and the cells were washed with 2 mM MgCl2 solution. After incubation with AP buffer (100 mM Tris−HCl, pH 9.5, 100 mM NaCl, and 10 mM MgCl2) for 15 min, the cells were treated in AP buffer containing 0.4 mg/mL of nitro-blue tetrazolium (NBT, Sigma) and 0.2 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) for 15 more minutes. To stop the reaction, the cells were exposed to 5 mM EDTA (pH 8.0) and fixed with 10% NBF for 1 h.
The differentiation into osteoblast was evaluated regarding ALP activity. The ALP activity was determined using an Alkaline Phosphatase Assay Kit (ab83369; Abcam, Cambridge, MA, USA). Briefly, the cell lysates were incubated with p-nitrophenyl phosphate (p-NPP) solution at RT for 1 h in the dark. After stopping the reaction, the optical density was measured at 405 nm using a SpectraMax M2/M2e Microplate Readers (Molecular Devices, San Jose, CA, USA).
The differentiation into osteoblast was evaluated regarding ALP activity. The ALP activity was determined using an Alkaline Phosphatase Assay Kit (ab83369; Abcam, Cambridge, MA, USA). Briefly, the cell lysates were incubated with p-nitrophenyl phosphate (p-NPP) solution at RT for 1 h in the dark. After stopping the reaction, the optical density was measured at 405 nm using a SpectraMax M2/M2e Microplate Readers (Molecular Devices, San Jose, CA, USA).
7
Solubility Determination of Roflumilast
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DMSO or NMP (50 μL) was added to 15 mg of RFB. The mixed samples were incubated for 1 day at room temperature (RT) using a rotary shaker (Barnstead/Thermolyne Model 415110 Labquake Shaker/rotator, Thermo Fisher Scientific, USA). The undissolved RFB in the solvent was removed by centrifugation (21,130 × g, RT, 5 min) (Eppendorf Centrifuge 5417R, Eppendorf Inc., Germany) and RFB concentration in the supernatant was analyzed by measuring the absorbance at 320 nm using a UV-vis spectrometer (SpectraMax M2/M2e microplate reader, Molecular Devices, CA, USA, with DeNovix DS-11 software version v 4.1.9, Wilmington, DE, USA). All experiments were performed in triplicate.
8
Quantitative Proteomics and Cell Viability Assays
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In-gel fluorescence was recorded using an ETTAN
Dige Imager (GE Healthcare). Chemiluminescence was recorded using
a LAS-3000 Imaging System (Fujifilm). Absorbance in 96-well plates
was measured using a SpectraMax M2/M2e Microplate Reader from Molecular
devices. Culture media and reagents were obtained from Sigma-Aldrich,
Gibco (Life technologies) and A&E Scientific (PAA). For quantitative
proteomics (SILAC), R10K8 and R0K0 DMEM media were purchased from
Dundee cell products, and the cell dissociation buffer (enzyme free,
PBS-based) was obtained from Gibco (Life Technologies). Dialyzed FBS
was obtained from Sigma-Aldrich. All buffers were filtered using a
0.2 μM filter to prevent any contamination. MTS assay was performed
as previously described.3 (link)
Dige Imager (GE Healthcare). Chemiluminescence was recorded using
a LAS-3000 Imaging System (Fujifilm). Absorbance in 96-well plates
was measured using a SpectraMax M2/M2e Microplate Reader from Molecular
devices. Culture media and reagents were obtained from Sigma-Aldrich,
Gibco (Life technologies) and A&E Scientific (PAA). For quantitative
proteomics (SILAC), R10K8 and R0K0 DMEM media were purchased from
Dundee cell products, and the cell dissociation buffer (enzyme free,
PBS-based) was obtained from Gibco (Life Technologies). Dialyzed FBS
was obtained from Sigma-Aldrich. All buffers were filtered using a
0.2 μM filter to prevent any contamination. MTS assay was performed
as previously described.3 (link)
9
Quantifying Complement Deposition on Klebsiella
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Indirect ELISA was used to compare complement C3/C3b and C5–9 (membrane attack complex) deposition on K. pneumoniae using serum from seropositive and seronegative donors. Protocols described by Cox et al. [17 (link)] were followed with modifications. Seeding of antigen was done as previously described. After overnight incubation at 4 °C, wells were washed 5× with PBST. Mouse monoclonal to C3/C3b, or Mouse monoclonal to C5b-9 secondary antibody (Abcam, Cambridge, MA, USA) were diluted in PBST following manufacturer recommendations, and 100 µL were added to each well. Plates were incubated at RT for 2 h, followed by 5× washes with PBST before adding Rat monoclonal to IgG2a HRP (Abcam) as tertiary antibody. After incubation at 25 °C for 1 h, the plate was washed 5× with PBST before adding 100 µL of ABTS Peroxidase Substrate (KPL, Gaithersburg, MD, USA) to each well. The ELISA reaction was stopped after 30 min with 100 µL 1% sodium dodecyl sulfate, and the optical density (OD) of the reactions was read at 405 nm with a SpectraMax M2/M2e Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
10
HeLa Cell Culture and Quantitative Proteomics
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Culture media and reagents for HeLa cell culture were obtained from Sigma-Aldrich, Gibco (Life Technologies), Iris Biotech, A&E Scientific (PAA).
Fluorescence on the gel was recorded using an ETTAN DIGE Imager (GE Healthcare). Chemiluminescence was recorded using a LAS-3000 Imaging System (Fujifilm). Absorbance in 96-well plates was measured using a SpectraMax M2/M2e Microplate Reader from Molecular Devices.
For quantitative proteomics (spike-in SILAC and SILAC), R10K8 and R0K0 Dulbecco's modified Eagle's medium (DMEM) media were purchased from Dundee Cell Products. Cell dissociation buffer (enzyme free, phosphate-buffered saline (PBS) based), obtained from Gibco (Life technologies) was used instead of trypsin to detach the cells before passaging. Dialysed fetal bovine serum (FBS) was obtained from Sigma-Aldrich. For proteomics, all buffers were filtered using a 0.2-μM filter. Low-binding tubes (Protein LoBind tubes, Eppendorf) were used to carry out the enrichment of NMT substrates for MS-based proteomics and to freeze-dry the digested peptides. Plasmid encoding C-terminally FLAG tagged Cul4B (aa1-590) was obtained from Addgene, TurboFect transfection reagent was from Thermo Scientific, ANTI-FLAG M2 Affinity Gel was from Sigma-Aldrich. N3-TAMRA was purchased from Life Technologies.
Fluorescence on the gel was recorded using an ETTAN DIGE Imager (GE Healthcare). Chemiluminescence was recorded using a LAS-3000 Imaging System (Fujifilm). Absorbance in 96-well plates was measured using a SpectraMax M2/M2e Microplate Reader from Molecular Devices.
For quantitative proteomics (spike-in SILAC and SILAC), R10K8 and R0K0 Dulbecco's modified Eagle's medium (DMEM) media were purchased from Dundee Cell Products. Cell dissociation buffer (enzyme free, phosphate-buffered saline (PBS) based), obtained from Gibco (Life technologies) was used instead of trypsin to detach the cells before passaging. Dialysed fetal bovine serum (FBS) was obtained from Sigma-Aldrich. For proteomics, all buffers were filtered using a 0.2-μM filter. Low-binding tubes (Protein LoBind tubes, Eppendorf) were used to carry out the enrichment of NMT substrates for MS-based proteomics and to freeze-dry the digested peptides. Plasmid encoding C-terminally FLAG tagged Cul4B (aa1-590) was obtained from Addgene, TurboFect transfection reagent was from Thermo Scientific, ANTI-FLAG M2 Affinity Gel was from Sigma-Aldrich. N3-TAMRA was purchased from Life Technologies.
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