Macrophages loaded with microparticles as described above with various particle incubation concentrations were plated at 62 500 cells per cm2 in 96 well plates in 100 μL DMEMb. Cells were put in a cell incubator and left for 24 or 48 h. A mixture containing 100 μL of DMEMb and 20 μL of alamarBlue® was added to the media in each well. The plate was incubated for 3.5 h. A volume of 110 μL of each well was transferred to black bottomed well plates to improve the sensitivity of the fluorescence measurement. Solution fluorescence was measured on a TECAN M220 Infinite Pro plate reader (λex = 550 nm, λem = 590 nm). Fluorescence from wells without cells was subtracted as baseline.
M220 infinite pro
Manufactured by Tecan
Sourced in Switzerland
The M220 Infinite Pro is a multimode microplate reader that provides high-performance detection capabilities for a wide range of assays. It is designed to accurately measure absorbance, fluorescence, and luminescence in various microplate formats.
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Lab products found in correlation
α tocopherol, by Merck Group (1 mentions)
Dspe peg2k amine, by Malvern Panalytical (1 mentions)
Tracecert, by Merck Group (1 mentions)
Empyrean powder diffractometer, by Malvern Panalytical (1 mentions)
Dspe peg2k amine, by Avanti Polar Lipids (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
4 protocols using m220 infinite pro
1
Microparticle Uptake in Macrophages
2
Camptothecin-PEG Nanocrystal Synthesis
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About 5 ml of 0.8 mg/ml of CPT (Sigma Aldrich) and 3.2 mg/ml DSPE PEG2K Amine (Avanti Polar Lipids) in DMSO solution were pipetted dropwise into a 120 ml water mixture containing 1% w/w alpha‐tocopherol (Sigma). The mixture was stirred at 800 rpm under constant ultrasonication at room temperature (22°C) for 1 hr. CPT + PEG nanocrystals formed at the boundary where DMSO diffused into the water. The CPT + PEG nanocrystals were then centrifuged three times at 20°C with milliQ water (18.2 MΩ•cm) at 3,500 rpm. The concentration of Camptothecin in CPT + PEG nanocrystals was determined by dissolving the nanocrystals in DMSO and reading the absorbance at 366 nm using a spectrophotometer (Tecan M220 Infinite Pro). The successful incorporation of DSPE PEG2K Amine was validated via X‐Ray diffraction of DSPE PEG2K Amine, CPT, and alpha‐tocopherol in their free powder form compared to the CPT + PEG construct (Panalytical Empyrean Powder Diffractometer). DSPE PEG2K Amine content was analyzed using an inductively coupled plasma (ICP) Atomic Emission Spectrometer (Thermo iCAP 6300 Model) the detection of Phosphorous signals using calibration curves prepared with a Phosphorous Standard for ICP (TraceCERT, 1,000 mg/L P in H2O, Sigma).
3
Quantifying E. coli Cell-Associated COEs
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E. coli cells at OD600 nm = 1.0 were stained in clear 96-well plates (BD Biosciences, San Jose, CA) at 20 °C for 1 hour in the dark with shaking. Total volume of each sample was 200 μL and samples were measured in triplicate. After centrifugation of the plate (3500 rpm, 4 minutes), 100 μL of supernatant was transferred to a clean well for UV-vis absorption with a Tecan M220 Infinite Pro plate reader (Tecan, Männedorf, Switzerland). Absorbance was measured at 420 nm for COE1 series and 380 nm for COE2 series molecules. Control samples with no cells were treated the same and their absorbance values represented the total COE from which the supernatant values were subtracted to give the amount associated with cells.
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E. coli cells at OD600 nm = 1.0 were stained in clear 96-well plates (BD Biosciences, San Jose, CA) at 20 °C for 1 hour in the dark with shaking. Total volume of each sample was 200 μL and samples were measured in triplicate. After centrifugation of the plate (3500 rpm, 4 minutes), 100 μL of supernatant was transferred to a clean well for UV-vis absorption with a Tecan M220 Infinite Pro plate reader (Tecan, Männedorf, Switzerland). Absorbance was measured at 420 nm for COE1 series and 380 nm for COE2 series molecules. Control samples with no cells were treated the same and their absorbance values represented the total COE from which the supernatant values were subtracted to give the amount associated with cells.
4
Fluorescence Spectroscopy of Tau-Heparin Interactions
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ThT fluorescence intensity was measured with a Tecan M220 Infinite Pro plate reader for data in Figure 2 and Supplementary Figures S1 , S2 , S4 , S20 and with a Biotek Synergy II for other data. A 20 μL sample volume was added to a Corning 384 Flat Black low volume well plate and covered with black vinyl electrical tape to prevent evaporation. For the Tecan plate reader, excitation wavelength of 450 nm (9 nm bandwidth) and emission wavelength of 484 nm (20 nm bandwidth) were used. Number of flashes was set to 25 and readings were taken from the bottom. For the Biotek plate reader, an excitation wavelength of 440 nm (30 nm bandwidth) and emission wavelength of 485 nm (20 nm bandwidth) were used. Number of flashes was set to 10 and readings were taken from the bottom. In the tau:heparin ratio study (Figures 2 , 5C and Supplementary Figures S1 , S3 ), the tau187 concentration was fixed to 100. For data in Figure 5 , the concentrations of tau187 50 μM. For clarity, every 60th time points were shown in the ThT kinetic curves. In parallel of all fluorescence reading, absorption at 500 nm was recorded to verify the absence of liquid droplets (phase separation) in the initial conditions. 20 μM ThT was used for all fluoerescence experiments.
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