Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

Piccinini, Filippo; Tesei, Anna; Arienti, Chiara; Bevilacqua, Alessandro

doi:10.1186/s12575-017-0056-3

Cited by 87 publications

(62 citation statements)

References 49 publications

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“…The Trypan Blue Exclusion assay is notorious for low accuracy, poor reproducibility and high variation between counts of the same preparation of cells, thought to be caused by non-specific binding of the TB dye to cellular artifacts [54]. Our optimized TB assay has coefficients of variation similar to, or lower than those reported previously [54] [57] [58]. In the present study, when preparations containing 625 granulosa cells were counted on four separate occasions, the intra-assay CoV of 7.6%, and the inter-assay CoV was 4%.…”

Section: Discussionmentioning

confidence: 99%

“…Piccinini F, Tesei A, Arienti C and Bevilacqua A [54] found no difference in viability when cells retrieved from two dimensional (2D) or three dimensional (3D) culture systems were assessed by TB. Cell viability and density measures varied by 5% and 20% respectively, with percent error in estimating cell numbers higher for 3D systems (21%) than for 2D systems (17%) [54]. Other studies reported coefficients of variation (CoV) for estimating cell numbers using TB from 4.5% [57] to 6.9% [58].…”

Section: Introductionmentioning

confidence: 99%

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Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

Asaduzzaman¹,

Gonzalez²,

Young³

2018

IJCM

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Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37˚C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ± 87) GC per antral follicle of which 82% ± 5% were viable. Culturing 312 -20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R 2 = 0.98) and estrogen concentration per well (R 2 = 0.92). 20,000 GC per well produced 143 ± 16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents. of 209 GCs per follicle (equivalent to a late secondary stage follicle [42], the CoVs for the TB assay were of the same order of magnitude as those reported by the manufacturers of commercially available EIA kits, and the CoVs for cell numbers corresponding to groups of three small primary follicles were within the ranges previously found acceptable for other cell-based assays [75]. From this we conclude that we developed an assay protocol with relatively high precision and accuracy. Although there are numerous reports of primary-derived human granulosa cells being cultured in vitro [76]-[82] and many instances in which GC obtained from the follicles of larger mammals have been cultured [83] [84] [85] [86] [87], as far as we know this is the first report describing the in vitro culture of GC obtained from the follicles of naturally cycling adult mice in a 96-well format. Cell viability assays that use the high throughput 96-well plate formats benefit from the application of control standard plots, in which known cell densities are related to optical density values [88] [89] [90]. Previously granulosa cells have been reported as having a doubling rate of 46.4 hours [67], hen...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

Asaduzzaman¹,

Gonzalez²,

Young³

2018

IJCM

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“…Using the appropriate dose for cell s' treatment is a very important issue, these doses should be chosen in a way that does not cause cell death and also have the maximum efficiency on cell differentiation (Piccinini et al, 2017). To determine the appropriate dose for the induction of differentiation, trypan blue assay was used; this assay detects the cell metabolic activity and viability (Piccinini et al, 2017). The results indicated that, the doses of fisetin that were less than 200 µg/ml had no significant effects on the proliferation of BMSCs.…”

Section: Discussionmentioning

confidence: 99%

The Osteogenic Differentiation of Mice Bone Marrow Mesenchymal Stem Cells with Fisetin Phytoestrogen

Kheiry¹,

Parivar²,

Baharara³

et al. 2017

JMBR

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Objective(s): The aim of this study was to evaluate the effects of fisetin to promote osteogenic differentiation in mice bone marrow mesenchymal stem cells (BMSCs). Materials and Methods:In this study cytotoxicity and viability of fisetin was measured by MTT assay. The differentiation effects of fisetin on BMSCs into osteoblast was assessed with alkaline phosphatase (ALP) activity measurement. Alizarin red staining and Real time PCR for osteoblast specific marker, Osteocalcin (OCN) ,Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), ERK and MAPK were investigated Results:The results showed that fisetin does not have toxicity effect on BMSCs and it causes cell proliferation; hence 200, 400 and 800 µg/ml of fisetin was selected for the assessment of differentiation progress. Alizarin red staining (ARS) showed that fisetin promotes osteogenic differentiation on BMSCs at 21st day; dependently also higher alkaline phosphates activity was observed in the treatment groups of 10 days culture, compared to the control groups. The evaluation of Real time -PCR result evaluated showed that OCN OPN, RUNX2,ERK and MAPK genes expressions were increased. Conclusion:The results of this method, showed that differentiation in bone marrow stem cells took place through p38 MAPK, and ERK1 gene activation.

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“…This method was done as demonstrated previously [42]. Briefly, cells were plated into 96 well plates (1×10 5…”

Section: Trypan Blue Dye Exclusion Assaymentioning

confidence: 99%

Anti-Proliferation, Pro-Apoptosis and Anti-Migration Effects of Ginkgolic Acid C13:0 Isolated from Ginkgo Biloba Exocarp in MCF-7 and 4T-1 Breast Cancer Cells

Zhou¹,

Jiang²,

Fu³

et al. 2018

Preprint

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Ginkgolic acids (GA) have been reported to exhibit anticancer properties, however, 15 the mechanisms remain unclear. This study aims to investigate the mechanisms of GA C13:0 16 that was isolated from Ginkgo biloba exocarp (GBE) for anti-proliferation, pro-apoptosis and 17anti-migration effects in human MCF-7 and mouse 4T-1 breast cancer cells. The cytotoxic 18 effect, apoptosis induction and migration inhibition were measured using MTT, TUNEL and 19Wound healing assays. The expression of mRNA and protein were determined using qPCR 20 and Western blot. Our results showed that no cytotoxicity was found at concentrations of 21 C13:0 below 100µM. The effects of GA C13:0 was further demonstrated by up-regulation of 22 the Bax/Bcl-2 apoptosis pathway and the expression of Apaf-1 protein in the mitochondria. In 23 addition, GA C13:0 also suppressed cell migration and epithelial to mesenchymal transition 24 (EMT) with the increase of E-cadherin expression accompanied by the decrease of Snail, 25 MMP-2, MMP-9 and Vimentin expression. Moreover, GA C13:0 induced cytochrome P450 26 (CYP) 1B1 expression in aryl hydrocarbon receptor (AhR) pathway. Notably, the 27 up-regulation of CYP1B1 also might play a pivotal regulatory role in mitochondrial and EMT 28 pathways in MCF-7 and 4T-1 cells. Our results may have implications for the development of 29 anticancer agents containing GA as functional additives. 30

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Cell Counting and Viability Assessment of 2D and 3D Cell Cultures: Expected Reliability of the Trypan Blue Assay

Cited by 87 publications

References 49 publications

Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

The Osteogenic Differentiation of Mice Bone Marrow Mesenchymal Stem Cells with Fisetin Phytoestrogen

Anti-Proliferation, Pro-Apoptosis and Anti-Migration Effects of Ginkgolic Acid C13:0 Isolated from Ginkgo Biloba Exocarp in MCF-7 and 4T-1 Breast Cancer Cells

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