Effects of serum protein and colloid on the alamarBlue assay in cell cultures

Goegan, Patrick; Johnson, Gerald R.; Vincent, Renaud

doi:10.1016/0887-2333(95)00004-r

Cited by 120 publications

(74 citation statements)

References 6 publications

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“…The optical density was immediately measured using a spectrophotometer (Sunrise, Tecan; Männedorf, Zurich, Switzerland) at wavelengths of 570 and 595 nm. The number of viable cells correlates with the magnitude of dye reduction [11,43,44] and it is expressed as a percentage of AlamarBlue reduction according to the manufacturer's protocol. However, each test was repeated at least three times in triplicate.…”

Section: Alamarblue Assaymentioning

confidence: 99%

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

Gloria

Russo

D’Amora

et al. 2013

J. R. Soc. Interface.

172

126

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In biomedicine, magnetic nanoparticles provide some attractive possibilities because they possess peculiar physical properties that permit their use in a wide range of applications. The concept of magnetic guidance basically spans from drug delivery and hyperthermia treatment of tumours, to tissue engineering, such as magneto-mechanical stimulation/activation of cell constructs and mechanosensitive ion channels, magnetic cell-seeding procedures, and controlled cell proliferation and differentiation. Accordingly, the aim of this study was to develop fully biodegradable and magnetic nanocomposite substrates for bone tissue engineering by embedding irondoped hydroxyapatite (FeHA) nanoparticles in a poly(1-caprolactone) (PCL) matrix. X-ray diffraction analyses enabled the demonstration that the phase composition and crystallinity of the magnetic FeHA were not affected by the process used to develop the nanocomposite substrates. The mechanical characterization performed through small punch tests has evidenced that inclusion of 10 per cent by weight of FeHA would represent an effective reinforcement. The inclusion of nanoparticles also improves the hydrophilicity of the substrates as evidenced by the lower values of water contact angle in comparison with those of neat PCL. The results from magnetic measurements confirmed the superparamagnetic character of the nanocomposite substrates, indicated by a very low coercive field, a saturation magnetization strictly proportional to the FeHA content and a strong history dependence in temperature sweeps. Regarding the biological performances, confocal laser scanning microscopy and AlamarBlue assay have provided qualitative and quantitative information on human mesenchymal stem cell adhesion and viability/proliferation, respectively, whereas the obtained ALP/DNA values have shown the ability of the nanocomposite substrates to support osteogenic differentiation.

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Section: Alamarblue Assaymentioning

confidence: 99%

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

Gloria

Russo

D’Amora

et al. 2013

J. R. Soc. Interface.

172

126

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“…Proliferation of cultures of precon¯uent SV cells treated with either 0, 5, 25 or 100 mM DHEA for 3 d was determined by counting cells on a haemocytometer and measuring the metabolic activity of the cells using the AlamarBlue procedure. 21 Freshly split SV cells were seeded at 5Â10 3 acm 2 in 12-well dishes for measuring cell proliferation via direct counting (n 6atrt) and by the AlamarBlue procedure (n 6atrt). To access metabolic activity, AlamarBlue (AccuMed International, Inc, Westlake, OH) was added to the test wells on day 3 of treatment, at a concentration of 1% of the total well volume.…”

Section: Isolation and Culture Of Sv Cells From Human Adipose Tissuementioning

confidence: 99%

Dehydroepiandrosterone alters the growth of stromal vascular cells from human adipose tissue

et al. 1999

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OBJECTIVE: The purpose of this study was to determine if the antiobesity actions of dehydroepiandrosterone (DHEA) observed in vivo are due to an in¯uence on the proliferation and differentiation of primary cultures of stromal-vascular (SV) cells isolated from human adipose tissue. DESIGN: SV cells were isolated from subcutaneous adipose tissue obtained from a young adult female undergoing elective liposuction. For the proliferation assay (Experiment 1), cultures were fed proliferation media containing 0, 5, 25 or 100 mM DHEA for 3 d. At the end of this treatment period, cultures were either prepared for counting or for determining their metabolic activity using the Alamar Blue staining procedure. For the differentiation assays (Experiment 2), cultures were fed differentiation media containing 0, 25 or 50 mM DHEA for 20 d. At the end of this treatment period, cultures were either prepared for lipid staining using Oil Red O or for marker enzyme analysis (glycerol-3-phosphate dehydrogenase activity; GPDH). To determine if the stimulatory effects of DHEA on SV cell differentiation were dependent on the presence of thiazolidinediones (Experiment 3), cultures of differentiating SV cells were incubated in the presence and absence of BRL 49653 and either 0, 25 or 50 mM DHEA. RESULTS: In Experiment 1, cultures treated with 25 and 100 mM DHEA had fewer cells than cultures treated with either 0 or 5 mM DHEA. Alamar Blue staining decreased as the level of DHEA in the cultures increased. In Experiment 2, cultures treated with DHEA had more lipid and GPDH activity than control cultures. In Experiment 3, cultures treated with BRL 49653 had more triglyceride than cultures treated without BRL 49653. Likewise, cultures treated with DHEA had more triglyceride than their non-DHEA controls. Regardless of the BRL status, cultures supplemented with DHEA had more triglyceride than control cultures. CONCLUSION: These data suggest that in cultures of SV cells from human adipose tissue, DHEA supplementation attenuates proliferation and enhances differentiation. These data support the hypothesis that DHEA directly attenuates preadipocyte proliferation in humans as we previously demonstrated in primary cultures of pig and rat SV cells and in cultures of 3T3-L1 preadipocytes. In contrast, DHEA stimulated the differentiation of human preadipocytes, which is contrary to its actions in differentiating cultures of preadipocytes from animals.

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“…Consequently, each individual sample in a dilution series has a different final concentration of serum. It is a documented feature of alamarBlue that the fluorescence signal can be quenched by the presence of serum (6). This quenching phenomenon was also observed in the HT-SBA and can result in an overestimation of titer.…”

Section: Determination and Optimization Of Assay Parameter Values (Imentioning

confidence: 89%

Development of an Automated, High-Throughput Bactericidal Assay That Measures Cellular Respiration as a Survival Readout for Neisseria meningitidis

Mak

Santos

Masterman

et al. 2011

Clin. Vaccine Immunol.

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Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.

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Effects of serum protein and colloid on the alamarBlue assay in cell cultures

Cited by 120 publications

References 6 publications

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

Magnetic poly(ε-caprolactone)/iron-doped hydroxyapatite nanocomposite substrates for advanced bone tissue engineering

Dehydroepiandrosterone alters the growth of stromal vascular cells from human adipose tissue

Development of an Automated, High-Throughput Bactericidal Assay That Measures Cellular Respiration as a Survival Readout for Neisseria meningitidis

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