Flow cytometry is used to identify and enumerate specific cell populations like stem cells in a product. Two methods determine cell viability - trypan blue dye exclusion identifies live and dead cells under a microscope, while flow cytometry identifies cells with damaged membranes allowing fluorescent compounds to insert into DNA. The product is also tested for blood type, total viable cell count using an analyzer, and sterility over 14 days using a microbial detection system.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
Comparison of Different Methods to Measure Cell ViabilityCreative-Bioarray
Cell viability assays are often used to screen collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. According to the number and type of cells used, choosing a proper assay method decided whether the expected outcome is valid or not.
Cell proliferation assays are used to monitor the dynamic growth of a cell population or to detect daughter cells in a growing population.
On the other hand, cell viability assays assess how healthy the cells are by measuring markers of cellular activity.
https://www.creative-bioarray.com/products/cell-viability,-proliferation-and-cytotoxicity-list-226.htm
The Main Advantage
The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher and slower pace.
Aquatic research
In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains.
This research started in 1981 when Clarice Yentsch used flow cytometry to measure the fluorescence in a red tide producing dinoflagellates
Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity, to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity,and to measure biogeochemical rates of multiple processes in the ocean
Cell Proliferation assay
Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments
Cell counting
Cell sorting
Determining cell characteristics and function
Detecting microorganisms
Biomarker detection
Protein engineering detection
Diagnosis of health disorders such as blood cancers
Flow cytometry can be used for cell cycle analysis to estimate the percentages of a cell population in the different phases of the cell cycle, or it can be used with other reagents to analyze just the S phase.
Why flow cytometry is ideal for cell cycle analysis
Live-cell cycle analysis stains—Vybrant DyeCycle stains
Classic DNA cell cycle stains such as Hoechst 33342 and DRAQ5 for cell cycle analysis, but most of these have limitations that have to be considered when using them in an experiment which is why the Invitrogen Vybrant DyeCycle stains for live-cell cycle analysis were developed.
Fixed-cell cycle analysis stains FxCycle reagents
We offer classic DNA cell cycle stains such as DAPI, PI, and 7-AAD for fixed cell cycle analysis, but these reagents do not cover the full spectrum of laser excitation available.
The FxCycle reagents offer options for the 405 nm (violet) and 633 nm (red) laser thereby increasing the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses such as immunophenotyping, apoptosis analysis, and dead cell discrimination.
Precise—Accurate cell cycle analysis in living cells
Safe—Low cytotoxicity for combining with additional live cell experiments
Cell sort compatible—Easily sort cells based on phase of the cell cycle
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
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During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
2. Flow Cytometry
A flow cytometer is used to determine the identify and size of specific cell populations in the product.
Example: CD34 Enumeration
The CD34 molecule is a cell surface antigen which exclusively identifies stem cells of the hematopoietic
system. The flow cytometeris used to enumerate this CD34 marker and identify the size of the stem cell
population in order to calculate the target dose.
3. Determining Cell Viability
Viability (proportion of living and dead cells) is an important indicator of the overall potency of the stem cell product.
Two methodologies are used to determine viability.
• Trypan Blue Dye Exclusion: Dead cells appear blue under the microscope as their damaged membranes allow
trypanbluedye to pass through, while live cells are colorless.
• Flow Cytometry: Fluorescent compounds penetrate dying and dead cell membranes and insert into the DNA.
The flow cytometer is able to accurately identify these dyed cells, and determine the viability of specific cell populations.
4. ABO / RH Typing
In order to help confirm identity, units are blood typed prior to processing.
5. Determining the Total Viable Nucleated Cell Count
An automated hematological analyzer is utilized to measure and differentiate cell types
in the stem cell product. This information is used to assess the processing efficacy and
quality of the product. The white blood cell count is often used as a release criteria, or
component of the cell dose calculation.
6. 14 Day Product Sterilities
An automated microbial detection system is used to monitor product
sterilities. Microorganisms present in the cultures release carbon
dioxide, triggering the instrument to alert the user that there is a positive
sample.
