Curcumin was loaded to SMA and FA-DABA-SMA polymers through physical entrapment. Briefly, excess curcumin was added to polymer stock solutions at 54 μm concentration. The solution was allowed to settle and was centrifuged at 4,000 rpm (2500× g) for 7 minutes to remove the remaining curcumin from the solution. Curcumin-loaded polymers were then diluted to four different concentrations of 0.3, 1, 3, and 10 μm. The curcumin-loaded SMA was characterized using Tecan M1000 Pro plate reader (Tecan Group Ltd, Männedorf, Switzerland) in 96-well microplates.
M1000 pro plate reader
Manufactured by Tecan
Sourced in Switzerland, United States
The Tecan M1000 Pro is a plate reader designed for high-performance absorbance, fluorescence, and luminescence detection. It features a wide wavelength range and high-precision optics to enable accurate measurements across a variety of assays and sample types.
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62b40peg, by PerkinElmer (2 mentions)
62inspeb, by PerkinElmer (2 mentions)
Tetramethyl benzene, by Merck Group (2 mentions)
Microtitration plates, by Corning (2 mentions)
Streptavidin hrp, by Abcam (2 mentions)
Polyclonal goat anti mouse igg2c, by Abcam (2 mentions)
A9044, by Merck Group (1 mentions)
Ez rich defined media, by Teknova (1 mentions)
Zetasizer nano zs90, by Malvern Panalytical (1 mentions)
Ht7700, by Hitachi (1 mentions)
Sense microplate reader, by Hidex (1 mentions)
Enduren, by Promega (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Pharm lyse solution, by BD (1 mentions)
Leucosep tube, by Greiner (1 mentions)
Nanosizer, by Malvern Panalytical (1 mentions)
Dpx 300 spectrometer, by Bruker (1 mentions)
35 protocols using m1000 pro plate reader
1
Curcumin-Loaded Polymer Characterization
2
Quantification of Protochlorophyllides in Arabidopsis
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Protochlorophyllides (Pchlides) were extracted and measured using published methods (Kolossov and Rebeiz, 2003 (link)) with modifications. Around 100 mg of etiolated Arabidopsis seedlings (7-d-old) were harvested under dim-green safe light, frozen and ground to fine powder. Two milliliters of 80% ice-cold acetone was added to each sample and the mixture was briefly homogenized. After centrifugation at 18,000 g for 10 min at 1 ⁰C, supernatant was split to 2 × 1 mL for Pchlides and protein extraction. Fully esterified tetrapyrroles were extracted from the acetone extracts with equal volume followed by 1/3 vol of hexane. Pchlides remained in the hexane-extracted acetone residue were used for fluorescence measurement with a TECAN M1000PRO plate reader (Tecan Group) and net fluorescence were determined as previously described (Rebeiz et al., 1975 (link)). Protein extraction was performed using 80% acetone and 10% TCA; protein concentration was used to normalize the net fluorescence of Pchlides.
3
SARS-CoV-2 Spike RBD IgG Antibody ELISA
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ELISA plates (Thermo Fisher #3455) were coated overnight at 4°C with 2 μg/mL of SARS-CoV-2 Spike RBD (Sino Biological #40592-V08B) in bicarbonate buffer (Sigma-Aldrich #88975) and blocked by 1% BSA in PBS with 0.1% Tween 20 (PBST) for 1 hr at room temperature. Serum samples were 3-fold serially diluted (in blocking buffer), starting at 1:100, and loaded to each well (100 μL). Following incubation at 37°C for 60 minutes, plates were washed 3 times with 1× PBS. Then 100 μL of diluted anti-mouse IgG-peroxidase rabbit antibody (Sigma-Aldrich #A9044, 1:10,000 dilution) was added per well and incubated at 37°C for 30 minutes. Plates were washed 3 times following incubation, and TMB substrates were added (100 μL/well) and incubated for 15-20 minutes at room temperature. Then, the ELISA stop buffer was added and the absorbance (450/630 nm) was measured with the TECAN infinite M1000 Pro plate reader. The endpoint IgG titers were defined as the highest dilution fold of sera to yield an absorbance greater than 2.1-fold of the background values (without serum but the secondary antibody was added) as previously described.27 (link) Antibody titer below the limit of detection was determined as half the limit of detection.
4
Evaluating CrCl3 Cytotoxicity with MTT Assay
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The effects of CrCl3 on cellular viability were assessed
using the MTT cell proliferation
assay.68 (link),69 (link) Briefly, 2 × 104 cells per
well were seeded onto Corning Scientific Costar 96-well polystyrene
flat bottom plates. After 24 h, the growth medium was aspirated and
replaced with new growth medium containing CrCl3. Sodium
dodecyl sulfate (0.05, 0.10, 0.15, or 0.20 mg/mL) was used as a positive
control for the assay.
After incubation for 1 h, the growth
medium was carefully aspirated and replaced with 100 μL of fresh
growth medium (absent of phenol red) and 10 μL of the MTT reagent.
After 6 h, the MTT solution was carefully aspirated and the formazan
crystals were dissolved with 50 μL of spectroscopic grade DMSO.
The absorbance at 540 nm was read by an M1000 PRO plate reader (Tecan,
Switzerland) following 1 s of shaking at 2 mm amp and 654 rpm. To
remove the background media effects, empty (blank) wells were also
treated according to the MTT protocol.
using the MTT cell proliferation
assay.68 (link),69 (link) Briefly, 2 × 104 cells per
well were seeded onto Corning Scientific Costar 96-well polystyrene
flat bottom plates. After 24 h, the growth medium was aspirated and
replaced with new growth medium containing CrCl3. Sodium
dodecyl sulfate (0.05, 0.10, 0.15, or 0.20 mg/mL) was used as a positive
control for the assay.
After incubation for 1 h, the growth
medium was carefully aspirated and replaced with 100 μL of fresh
growth medium (absent of phenol red) and 10 μL of the MTT reagent.
After 6 h, the MTT solution was carefully aspirated and the formazan
crystals were dissolved with 50 μL of spectroscopic grade DMSO.
The absorbance at 540 nm was read by an M1000 PRO plate reader (Tecan,
Switzerland) following 1 s of shaking at 2 mm amp and 654 rpm. To
remove the background media effects, empty (blank) wells were also
treated according to the MTT protocol.
5
Cell Proliferation Assay via GFP Fluorescence
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To determine cell proliferation rate using a fluorescence plate reader, TNBC cells were stably transfected with GFP (pRetroQ‐AcGFP1‐N1). Transfected cells were selected with puromycin (BT‐20 at 1.5 μg/ml, 468 at 0.5 μg/ml, and 231 at 2 μg/ml). Cells were selected until a parallel non‐transformed plate exposed to puromycin was completely dead. The selected population was subsequently sorted by FACS to collect cells with similar levels of GFP fluorescence. For coculture experiments, fibroblast cell lines were plated at an 8:1, 4:1, 2:1, 1:1, and 1:2 ratio to cancer cells in a Greiner 96‐well plate in 100 μl of FluoroBrite media and allowed to adhere for 3 h. Following adherence of fibroblast cells, TNBC cells constitutively expressing GFP were plated at a concentration of 10,000 cells per 100 μl of FluoroBrite media and allowed to adhere overnight. Cell measurements were measured every 24 h for 96 h using a Tecan M1000 Pro Plate reader.
6
3D Culture and Drug Response Assay for TNBC
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Culturing of TNBC cells in 3D colonies was performed using the “3D on top” method developed by Bissell and colleagues (Lee et al, 2007 ). Briefly, a thick layer of cold Matrigel (corning cat#356235) was applied to the bottom of 96 well plates, which were subsequently heated to 37°C for 30 min to promote solidification. Cancer cells were plated at a concentration of 10,000 cells in 100 μl complete media + 2% Matrigel and were grown for 72 h to induce 3D colony formation. After 72 h of growth, the media were aspirated and media containing drug + 2% Matrigel were added to each well. At 72 h post‐drug addition, cell viability was measured by adding 100 μl of CellTiter‐Glo reagent to each well. The CellTiter‐Glo assay was performed according to the manufacturer's directions, and luminescence was read using a Tecan M1000 Pro Plate Reader.
7
Quantifying Cardiac Creatine Kinase Activity
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After cervical dislocation, blood was collected from the heart, placed into cryovials, centrifuged at 4000 rpm for 10 min at 4 °C, and serum collected. A colorimetric creatine kinase activity assay kit was used to determine CK-MM levels (Abcam, Melbourne, Vic. Australia Cat. No.: ab155901). To determine optimal diluent concentration for the assay, a standard curve was created and then samples were run in duplicate and were measured at OD 450 nm on a Tecan M1000 Pro Plate Reader in a kinetic mode, every 1 min for 40 min at 37 °C.
8
Spectrophotometric Assay for TPI Activity
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TPI activity was determined using a spectrophotometric assay as previously described.29 , 38 Measurements were performed at 2‐3 different protein concentrations per tissue lysate and repeated independently three times with freshly dissected tissues from 20‐week‐old male mice. NADH loss was measured using a Tecan M1000 PRO plate reader. TPI activity was calculated at the point of maximal reaction rate and normalized to lysate‐free background activity.
9
Hemin-Catalyzed Oxidation of ABTS
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Hemin was dissolved in DMSO to obtain a solution at 1 mM concentration and stored in the dark at –20°C. ABTS2−and H2O2were freshly prepared in water at the desired concentration for use. DNA samples were folded by heating at 95°C for 5 min in the corresponding buffer and left to cool at room temperature for at least two hours. 5 μl of a fresh dilution of Hemin at 60 μM (10% DMSO) were then added to reach a final Hemin concentration of 6 μM and the solutions were left to stand at room temperature (25°C) for 30 min. 5 μl ABTS2− at 5 mM was then added to each sample to a final concentration of 500 μM, and basal absorbance was measured. To initiate the oxidation reaction, H2O2 was added to a final concentration of 50 μM, followed by quick mixing. 15 min later, the absorbance at 420 nm of the oxidized product ABTS•− was monitored by a TECAN M1000 pro plate reader (France). The final sample contained 3 μM DNA, 6 μM Hemin, 1% DMSO, 500 μM ABTS2− and 50 μM H2O2 in a total volume of 50 μl.
10
Membrane Interactions of P0ct Protein
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For turbidimetric measurements, SUVs of 0.5 mM DOPC:DOPG (1:1) and DMPC:DMPG (1:1), both with and without supplemented 10% (w/w) cholesterol, were mixed with 0.5–10 µM P0ct in duplicate. Light scattering was recorded at 450 nm for 10 min at 25 °C using a Tecan M1000Pro plate reader. The results were analyzed after the observed optical density per time had stabilized.
SAXD experiments were performed to investigate any repetitive structures in turbid samples. 10 and 20 µM P0ct was mixed with SUVs of 1–3 mM DMPC:DMPG (1:1) in HBS at ambient temperature and exposed at 25 °C on the EMBL P12 BioSAXS beamline, DESY (Hamburg, Germany). A HBS buffer reference was subtracted from the data. Lipid samples without added P0ct were devoid of Bragg peaks. The peak positions of momentum transfer, s, in P0ct-lipid samples were used to calculate mean real-space repeat distances, d, in proteolipid structures, using the equation
SAXD experiments were performed to investigate any repetitive structures in turbid samples. 10 and 20 µM P0ct was mixed with SUVs of 1–3 mM DMPC:DMPG (1:1) in HBS at ambient temperature and exposed at 25 °C on the EMBL P12 BioSAXS beamline, DESY (Hamburg, Germany). A HBS buffer reference was subtracted from the data. Lipid samples without added P0ct were devoid of Bragg peaks. The peak positions of momentum transfer, s, in P0ct-lipid samples were used to calculate mean real-space repeat distances, d, in proteolipid structures, using the equation
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