Cell proliferation was determined by Alamar‐Blue colorimetric assay (Invitrogen, CA, USA) as previously reported 50 . Briefly, 3.000–5.000 cells/well (3–5 individual experiments, 4 wells/treatment) were seeded in 96‐well plates. Then, cells were 24 hrs‐starved (serum‐free‐medium) and proliferation rate measured, every 24 hrs, for the following 4 days. On the day of measurement, cells were incubated for 3 hrs in 10% Alamar‐Blue/RPMI without FBS and then Alamar‐Blue reduction was measured in FlexStation III (Molecular Devices, Madrid, Spain), exciting at 560 nm and reading at 590 nm. Medium with Alamar‐Blue was replaced with fresh medium with treatments (insulin or IGF1) immediately after each measurement. Results are expressed as percentage versus control (cell without treatment).
Alamarblue colorimetric assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlamarBlue® is a colorimetric assay used to quantitatively measure cell viability and proliferation. It utilizes the active ingredient resazurin, which is converted to the fluorescent product resorufin by metabolically active cells.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (3 mentions)
Alamar blue dye, by Thermo Fisher Scientific (3 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (3 mentions)
Celltiter96 dye, by Promega (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Phase contrast microscopy, by Leica (1 mentions)
Biotek gen5, by Agilent Technologies (1 mentions)
Aqueous non radioactive cell proliferation assay, by Promega (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
9 protocols using alamarblue colorimetric assay
1
Quantifying Cell Proliferation via Alamar Blue
2
Scaffold-Mediated Cell Proliferation and Viability
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Cell proliferation and viability cultured using the Mg-coated Ti6Al4V scaffold extraction (Mg) and bare Ti6Al4V scaffold extraction (Ti), as well as pure complete medium (pM), were evaluated at 1, 4, 7, and 14 days, respectively, using an alamarBlue colorimetric assay (Invitrogen, Carlsbad, CA, USA). 3 × 103 (MC3T3-E1 cells) and 2 × 103 (HUVECs) cells per well were seeded on 96-well plates. Each well was added with 10 μL of 10% (v/v) alamarBlue solution and cultured for 3 h. A microplate reader was applied to test the absorbance of the culture medium at 570/600 nm in triplicate.
Following the manufacturer's guidance, cell viability was further assessed by the Calcein-AM/PI Double Stain Kit. MC3T3-E1 cells and HUVECs cultured with pM, Ti and Mg were resuspended and seeded on 24-well plates at a density of 5 × 103 and 4 × 103 cells per well, respectively. After 1-week cultivation, the cells were stained with calcein AM (acetoxymethyl) and PI (propidium iodide) for living and dead cells, respectively. Cells were imaged using 490 nm wavelength excitation for green living cells and 545 nm for red dead cells by a confocal laser scanning microscope.
Following the manufacturer's guidance, cell viability was further assessed by the Calcein-AM/PI Double Stain Kit. MC3T3-E1 cells and HUVECs cultured with pM, Ti and Mg were resuspended and seeded on 24-well plates at a density of 5 × 103 and 4 × 103 cells per well, respectively. After 1-week cultivation, the cells were stained with calcein AM (acetoxymethyl) and PI (propidium iodide) for living and dead cells, respectively. Cells were imaged using 490 nm wavelength excitation for green living cells and 545 nm for red dead cells by a confocal laser scanning microscope.
3
Splenocyte Proliferation Assay Protocol
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Splenocytes obtained from aGKE-treated and control mice were suspended in RPMI-1640
medium containing 1U penicillin/ml, 100 μg streptomycin/ml, and 10% fetal calf serum (FCS)
(Sigma) at the concentration of 4x105 (link)
cells/100 μL. Concanavalin A (ConA) (Sigma) was added at 3 μg/mL, and the cells
were cultured for 72 h at 37°C under 5% CO2. Proliferation was quantified using
an Alamar Blue colorimetric assay (Invitrogen, Grand Island, NY), and the optical
densities measured following the manufacturer’s instructions by the ELISA plate reader
(GeneMate, Kaysville, UT), as described previously.4
medium containing 1U penicillin/ml, 100 μg streptomycin/ml, and 10% fetal calf serum (FCS)
(Sigma) at the concentration of 4x105 (link)
cells/100 μL. Concanavalin A (ConA) (Sigma) was added at 3 μg/mL, and the cells
were cultured for 72 h at 37°C under 5% CO2. Proliferation was quantified using
an Alamar Blue colorimetric assay (Invitrogen, Grand Island, NY), and the optical
densities measured following the manufacturer’s instructions by the ELISA plate reader
(GeneMate, Kaysville, UT), as described previously.4
4
Cellular Proliferation and Viability Assay
5
Viability and Proliferation of Cells from 3D Hydrogels
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Cells from 3D hydrogels (Gtn-HPA or MCh) were recovered via hydrogels digestion in the presence of 1 mg/mL collagenase G/H mix (1:10; Abiel, Palermo, Italy) for 60 min at 37 °C. Viability assay from 3D hydrogel or from 2D condition cells was assessed using Trypan Blue exclusion test. The cells were washed with PBS and counted in the Burker chamber. The cells recovered after gel digestion were seeded on a plate and their proliferative capability was monitored over time using the Alamar-Blue colorimetric assay (Thermo Scientific, Foster City, CA, USA) according to the manufacturer’s recommendations. Cells were incubated at different times, with an Alamar-Blue reagent solution (1:10) in their complete medium for 2 h in a humidified incubator (37 °C; 5% CO2). The increase in fluorescence was evaluated by a microplate reader (Synergy HT, Biotek, Winooski, VT, USA) using excitation/emission wavelengths of 530/590 nm. The standard curve was obtained through the use of known cell concentrations. Cells were stained with Coomassie Brilliant Blue G (0.8% Coomassie Brilliant Blue G-250 in Methanol/distilled water/acetic acid 5:5:1) for 10 min, then their morphology was assessed by phase-contrast microscopy (Leica).
6
Assessing Neuroblastoma PDX Cell Response
7
Assessing Tumor Cell Viability and Proliferation
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Following tumor dissociation, cells were suspended in NB media and counted. To assess cell survival, alamarBlue® colorimetric assay (Thermo Fisher Scientific) was utilized. Cells (1.5 × 104) were plated on 96-well plates and treated the following day with PF or Y15 in increasing concentrations, or an equivalent concentration of vehicle (DMSO). After 24 hours, 10 μL of alamarBlue® dye (Thermo Fisher Scientific) was added and absorbance was read at 570 nm and 600 nm on a microplate reader (Epoch Microplate Spectrophotometer, BioTek Instruments, Winooski, VT). Proliferation was evaluated using the colorimetric CellTiter96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Cells (5 × 103) were plated and treated the following day with vehicle, PF, or Y15 in increasing concentrations. CellTiter96® dye (10 μL, Promega) was added 24 hours later and absorbance read at 490 nm. Results in survival and proliferation from at least three biologic replicates were reported as fold change ± SEM.
8
Cell Viability in Electrospun Scaffolds
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At different times (1, 7, 14 and 21 days) from cell seeding, the viability and cell proliferation rate for each type of electrospinning device were evaluated using alamarBlue colorimetric assay (Thermo Scientific, Foster City, CA, USA) according to the manufacturer’s recommendations. Cells grown on each type of scaffold were incubated with Alamar-Blue reagent solution (10% in culture medium) in a complete medium for 2 h in a humidified incubator (37 °C; 5% CO2).
Fluorescence intensity (λexc 530/25 nm and λemm 590/35 nm expressed as relative fluorescent units (RFU)), which changes according to the degree of cell viability, was evaluated through a microplate reader (Synergy HT, Biotek, Winooski, VT, USA). The fluorescence values of each type of scaffold were normalized against the same type of unseeded device, used as a blank. The assay was performed in quadruplicate for each scaffold formulation and repeated three times. The cell viability was expressed as a percentage normalized to PLA or P-PLA scaffolds at time 1 (t1).
Fluorescence intensity (λexc 530/25 nm and λemm 590/35 nm expressed as relative fluorescent units (RFU)), which changes according to the degree of cell viability, was evaluated through a microplate reader (Synergy HT, Biotek, Winooski, VT, USA). The fluorescence values of each type of scaffold were normalized against the same type of unseeded device, used as a blank. The assay was performed in quadruplicate for each scaffold formulation and repeated three times. The cell viability was expressed as a percentage normalized to PLA or P-PLA scaffolds at time 1 (t1).
9
Crizotinib and Sunitinib Cytotoxicity Assay
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COA109 cells were suspended in Accutase (Fischer Scientific), incubated at 37 °C for 10 min, re-suspended in NB media, and counted. Cells (1.5 × 104) were plated in 96-well plates and treated with increasing doses of crizotinib (0, 0.5, 1, 2.5, 5, 10, 20 µm), sunitinib (0, 2.5, 5, 10 µm), or an equivalent concentration of dimethyl sulfoxide (DMSO) for a vehicle control. After 24, 48, and 72 h of treatment, an alamarBlue® colorimetric assay (Thermo Fisher Scientific) was used to assess viability. AlamarBlue® dye (10 µL, Thermo Fisher Scientific) was added and absorbance was read at 570 nm and 600 nm on a microplate reader (Epoch Microplate Spectrophotometer, BioTek Instruments, Winooski, VT). To assess proliferation, cells (5 × 103) were plated in 96-well plates and treated with control, crizotinib, or sunitinib in increasing concentrations (as above) for 24, 48, and 72 h and the colorimetric CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (10 µL, Promega, Madison, WI) was utilized. Absorbance was read at 490 nm with a microplate reader. Cell viability and proliferation from at least three biologic replicates were reported as fold change ± standard error of the mean (SEM).
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