A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry

Chan, Leo Li‐Ying; Smith, Tim; Kumph, Kendra A.; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

doi:10.1007/s10616-016-0015-x

Cited by 56 publications

(36 citation statements)

References 20 publications

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“…The manual method had a higher CV%, from 10% to 23%. The obtained results are in agreement with the results obtained by other authors using the manual method [11,17,30,31]. A comparison of the obtained linear interval and CV of Easycounter YC with other similar image cytometers showed that they have a similar CV, but the optimal interval is different for each different device: 1 Â 10 5 -2 Â 10 6 cells/mL with CV 3-10% for NucleoCounter YC-100 (company ChemoMetec) and 4 Â 10 6 -6.6 Â 10 7 cells/mL with CV 4-13% for Cellometer Auto X4 (10Â) (Nexcelom Bioscence) [11,23,28,31].…”

Section: Cell Count Linearity and Reliabilitysupporting

confidence: 92%

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Atanasova

Yordanova

Nenkova

et al. 2019

Biotechnology & Biotechnological Equipment

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This study presents an automated, image-based cytometry method for determination of total counts and viability of yeast cells by using a newly synthesized DNA fluorescent dye PO-TEDM-1 and a new Easycounter YC instrument. The synthesized polycationic asymmetric monomethine cyanine dye PO-TEDM-1 penetrates only into dead cells. The new fluorescent dye has high nucleic acid sensitivity and rapid interaction kinetics. The optimal concentration of the fluorescent dye for staining dead cells was 1 mg mL À1. The Easycounter YC system was used to determine the total cell count and viability of Saccharomyces carlsbergensis. The actual viability measured using the proposed method significantly correlated with the theoretical viability (R 2 of 0.9988). The optimal linear interval was from 1 Â 10 5 to 1 Â 10 7 cells mL À1. The coefficient of variation with Easycounter YC in the optimal range was 2.5-4%, whereas that of the manual hemocytometry method, in the same range was higher, 15-23%. We tested the procedure in a study of the total cell count and viability of yeast cells from the propagators in beer production as a function of the dilution. The proposed method can be used in assays involving simple cell counting and quality assurance in sample bioprocessing.

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Section: Cell Count Linearity and Reliabilitysupporting

confidence: 92%

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Atanasova

Yordanova

Nenkova

et al. 2019

Biotechnology & Biotechnological Equipment

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“…For example, when Acridine Orange and Propidium Iodide (AO/PI) are used for staining cells, AO permeates both live and dead nucleated cells, whereas PI enters only dead nucleated cells. As a result, all live nucleated cells fluoresce green and all dead nucleated cells fluoresce red, due to a phenomenon called as Forster resonance energy transfer or FRET [20]. RBCs are specifically not stained by AO/PI due to their lack of nucleus[21].…”

Section: Discussionmentioning

confidence: 99%

An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples

Hanamsagar

Reizis

Chamberlain

et al. 2019

Preprint

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Establishing clinically relevant single-cell (SC) transcriptomic workflows from cryopreserved tissue is essential to move this emerging immune monitoring technology from the bench to the bedside. Improper sample preparation leads to detrimental cascades, resulting in loss of precious time, money and finally compromised data. There is an urgent need to establish protocols specifically designed to overcome the inevitable variations in sample quality resulting from uncontrollable factors in a clinical setting. Here, we explore sample preparation techniques relevant to a range of clinically relevant scenarios, where SC gene expression and repertoire analysis are applied to a cryopreserved sample derived from a small amount of blood, with unknown or partially known preservation history. We compare a total of ten cell-counting, viability-improvement, and lymphocyte-enrichment methods to highlight a number of unexpected findings. Trypan blue-based automated counters, typically recommended for single-cell sample quantitation, consistently overestimate viability. Advanced sample clean-up procedures significantly impact total cell yield, while only modestly increasing viability. Finally, while pre-enrichment of B cells from whole peripheral blood mononuclear cells (PBMCs) results in the most reliable BCR repertoire data, comparable T-cell enrichment strategies distort the ratio of CD4+ and CD8+ cells. Furthermore, we provide high-resolution analysis of gene expression and clonotype repertoire of different B cell subtypes. Together these observations provide both qualitative and quantitative sample preparation guidelines that increase the chances of obtaining high-quality single-cell transcriptomic and repertoire data from human PBMCs in a variety of clinical settings.

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“…Previously, the Celigo Image Cytometer has been utilized to perform cell‐based assays in microplates by capturing and analyzing whole well images . In this work, the Celigo Image Cytometer was used to rapidly count sorted single cells in Greiner 96‐well half area plates in order to determine the sorting efficiency of the FACS.…”

Section: Methodsmentioning

confidence: 99%

A rapid single cell sorting verification method using plate‐based image cytometry

Zigon

Purseglove²,

Toxavidis

et al. 2018

Cytometry Pt A

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Single cell sorting is commonly used for ensuring monoclonality and producing homogenous target cell populations. Current single cell verification methods involve manually confirming the existence of single cells or colonies in a well using a standard light microscope. However, the manual verification method is time-consuming and highly tedious, which prompts a need for an accurate and rapid detection method for verifying single cell sorting capability. Here, we demonstrate a rapid single cell sorting verification method using the Celigo Image Cytometer. Calcein AM-stained Jurkat cells and fluorescent beads are sorted into 96-well half area microplates using the MoFlo Astrios EQ. Whole well bright field and fluorescent images are acquired and analyzed using the image cytometer in less than 8 min. The proposed single cell verification detection method in multi-well microplates can allow for quick optimization of FACS instruments at flow core laboratories, as well as improvement of downstream biological assays by accurately confirming the presence of single cells in each well.

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A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry

Cited by 56 publications

References 20 publications

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples

A rapid single cell sorting verification method using plate‐based image cytometry

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