This document describes the methods for conducting an in vitro chromosomal aberration test. White blood cells are cultured from human donors and exposed to test substances like cyclophosphamide. Cells are harvested at different timepoints throughout the cell cycle. Slides are prepared, stained, and analyzed under a microscope to identify chromosomal abnormalities like deletions, duplications, inversions, and translocations. Acceptance criteria include having homogenous results between replicates, aberration frequencies within historical controls, and a concentration-dependent response to a test substance. A conclusion on the potential of a substance to induce chromosomal aberrations is based on statistical analyses.
Alternative methods to animals testing are the development and implementation of test method that avoid use of live animals or use of less animals in method.
The council directive on protection of animals used for experiments and scientific purpose in article 23
“The commission and member states should encourage
research into development and validation of alternative methods which could provide the same level of information as that obtained in experiment using animals but which involves less animal”.
Alternative methods able to do:
Reduce Refine Replace
collectively called as “The 3Rs Principle”.
Needs for alternative methods
Because in laboratory animals may be:
Poisoned.
Deprived of food water and sleep.
Applied with skin and eye irritants.
Subjected to psychological stress.
Deliberately infected with the infected disease.
Alternative methods to animals testing are the development and implementation of test method that avoid use of live animals or use of less animals in method.
The council directive on protection of animals used for experiments and scientific purpose in article 23
“The commission and member states should encourage
research into development and validation of alternative methods which could provide the same level of information as that obtained in experiment using animals but which involves less animal”.
Alternative methods able to do:
Reduce Refine Replace
collectively called as “The 3Rs Principle”.
Needs for alternative methods
Because in laboratory animals may be:
Poisoned.
Deprived of food water and sleep.
Applied with skin and eye irritants.
Subjected to psychological stress.
Deliberately infected with the infected disease.
Acute eye irritation test as per OECD guidelinesmadhvi Chaubey
toxicological testing studies as per OECD guidline.
Toxicology is the branch of biology, chemistry and medicine concerned with the study of the adverse effects of chemicals on living organisms.
As per OECD test no. 405 : acute eye irritation test should be done as according to the procedure mentioned under guideline's section.
safety pharmacology is the branch of pharmacology specializing in detecting and investigating potential undesirable pharmacodynamic effects of a new chemical on physiological functions .
the content of this presentation is as follows
- introduction
- definition
- history
- ICH - guidelines
- refrences
This presentation enlists all the studies which are required before submission of IND. It include IND introduction , time period of study ,flowchart showing preclinical studies...
The basic aspects of drug discovery starts from target discovery and validation further going to lead identification and optimization. In this particular slide discussion is regarding the target discovery and the tools that have been utilized in this process.
Safety pharmacology is a branch of pharmacology with its aim to predict the potential clinical risk profile of new chemical entities (NCEs).
It has the ability to predict the potential off-target drug effects on major organ systems which are associated with exposure in the therapeutic range and above.
As an essential part of the spectrum of drug discovery and development, safety pharmacology studies are generally conducted to determine the relative drug effect on main organs, including respiratory system, central nervous system, and cardiovascular system.Safety pharmacology is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials.
SP studies are described in the international conference on harmonization (ICH) S7A and S7B Guidelines.
Role of nuclicacid microarray &protein micro array for drug discovery processmohamed abusalih
role of nuclic acid microarray and protein microarray for drug discovery process
1.introduction about microarray technique and genomics
2.process of drug discovery
3.microarray techiques
4.microarray analysis in drug discovery
5.steps involved in the micro array analysis
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
Acute eye irritation test as per OECD guidelinesmadhvi Chaubey
toxicological testing studies as per OECD guidline.
Toxicology is the branch of biology, chemistry and medicine concerned with the study of the adverse effects of chemicals on living organisms.
As per OECD test no. 405 : acute eye irritation test should be done as according to the procedure mentioned under guideline's section.
safety pharmacology is the branch of pharmacology specializing in detecting and investigating potential undesirable pharmacodynamic effects of a new chemical on physiological functions .
the content of this presentation is as follows
- introduction
- definition
- history
- ICH - guidelines
- refrences
This presentation enlists all the studies which are required before submission of IND. It include IND introduction , time period of study ,flowchart showing preclinical studies...
The basic aspects of drug discovery starts from target discovery and validation further going to lead identification and optimization. In this particular slide discussion is regarding the target discovery and the tools that have been utilized in this process.
Safety pharmacology is a branch of pharmacology with its aim to predict the potential clinical risk profile of new chemical entities (NCEs).
It has the ability to predict the potential off-target drug effects on major organ systems which are associated with exposure in the therapeutic range and above.
As an essential part of the spectrum of drug discovery and development, safety pharmacology studies are generally conducted to determine the relative drug effect on main organs, including respiratory system, central nervous system, and cardiovascular system.Safety pharmacology is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials.
SP studies are described in the international conference on harmonization (ICH) S7A and S7B Guidelines.
Role of nuclicacid microarray &protein micro array for drug discovery processmohamed abusalih
role of nuclic acid microarray and protein microarray for drug discovery process
1.introduction about microarray technique and genomics
2.process of drug discovery
3.microarray techiques
4.microarray analysis in drug discovery
5.steps involved in the micro array analysis
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
Prepared in July 2015
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
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
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
2. INTRODUCTION
• The adverse health effects caused by the Geno toxins in vivo are of
concern. The effects of exposure to relatively low concentrations of Geno
toxic substances may not be immediately apparent, this means that there
are no warning signs so that exposure can be avoided
• The short term in vitro mammalian cell chromosome aberrations test is
used to assess the potential Genotoxic hazard of test substances
• Abnormality in Chromosome can be of following Types
1. Deletion
2. Duplication
3. Inversion
4. translocation
30-04-2023 2
3. METHODS
• White Blood Cell Culture
• Dosing Formulation: Cyclophosphamide(treat cancer), anhydrous
dimethyl sulphoxide as a vehicle, S9 fraction
• Sampling, Slide preparation and Staining:
1. KCl in water(0.56%)
2. Methanol : Glacial acetic Acid(3:1 v/v)
3. Microscopic Slides
• Conduct of Test: High quality metaphase preparation is preferred, use
of a trained and experienced Observer
30-04-2023 3
4. METHODS
• Study Design: Culture initiation
Add WBC sample to culture medium including PHA and incubate(37±1°C)
for 48 Hrs, cells mostly in G0 of cell cycle
Start of Exposure: 0hrs
Add dosing formulations dimethyl sulphoxide & incubate for 3Hrs, cells at
all stages of cell cycle
End of Exposure: 3 hrs
Remove dosing Formulation. Incubate for 15Hrs. Cells should have
undergone S phase since 0 Hr
30-04-2023 4
5. Study Design
Metaphase Arrest: 18 hrs
Add Colcemid. Incubate for 2 hrs. Cells should be at first M phase since 0 hrs
Harvest: 20 hrs
Hypotonic to swell cells, fixative cell suspension on slides, stain and cover slip
30-04-2023 5
6. White Blood Culture
• Blood is to be taken by addition of Heparin tubes to avoid clotting for Blood
collection
• The sample is been then incubated at 37°C±1°C throughout with gentle mixing of
the cultures. A humidified atmosphere of 5% CO2 in air may be introduced at the
culture
• Lymphocytes are grown as suspension cultures in sterile, disposable centrifuge
tubes
• A healthy donor who do not smoke and willing to donate blood on a regular
basis is selected , absence of viral diseases such as hepatitis and HIV are
important both for protection of operators and for the success of the assay
• The Lymphocytes are tested for a good response to PHA giving an acceptable
mitotic index(MI) and AGT.
30-04-2023 6
7. WHITE BLOOD CULTURE
• The Lymphocytes should have a normal karyotype and exhibit levels of
chromosome aberrations that falls within a normal range with references to the
historical negative control database
• Prior to vein puncture, it should be ascertained that the potential donor has not
knowingly been exposed to ionizing radiation, hazardous chemicals or has
suffered from viral infections in the 2 weeks period before giving blood
• Heparinised blood, 0.4 ml is added to the warmed supplemented RPMI1640
medium so that the final volume following addition of S-9 mix/KCl and the dosing
formulation is 10ml
• These suspension cultures are incubated for approximately 48 hrs
• Even small but prolonged reductions in incubator temperature can affect the AGT
and this may not be obvious as it is likely to be measured peroidically
30-04-2023 7
8. PREPARATION, ADDITION & REMOVAL OF DOSING
FORMULATION
• Add approximately 10 mg CPA and add sufficient amount of DMSO to prepare the
stock solution that is 100x strong
• Prepare a alternative stock solution that is 10x strong and dissolve in water for
injection then sterilize to avoid microbial contamination
• The stock solution is diluted to provide six concentration of CPA , in the presence
of S-9 mix it
• The S-9 fraction is thawed just before required and the S-9 mixture is prepared
• The CPA dosing formulation(0.1 ml) is added to each culture to achieve the
required final concentration of CPA in a total of 10ml, culture exposed to CPA in
absence of S-9 receives 0.5 ml of KCl
• Incubation is continued for 3 hrs
30-04-2023 8
9. PREPARATION, ADDITION & REMOVAL OF DOSING
FORMULATION
• Culture tubes are centrifuged at approximately 300x g for 10 mins, the
supernatant is carefully removed and cells resuspended in 10 ml RPMI medium
without supplements, prewarmed to 37°C. This process is repeated so that there
have been two changes of medium, after the third centrifugation, the cells are
resuspended in 10ml complete RPMI medium, prewarmed at 37°C and
incubation is continued
30-04-2023 9
10. SAMPLING, SLIDE PREPARATION AND STAINING
• Colcemid are added 2-3 hrs before sampling to arrest dividing cells at metaphase
• At the sampling time, cultures are centrifuged at 300x g for 10 mins, the
supernatant carefully removed and cells resuspended in 4ml warmed KCl at 37°C
for 15 mins to allow cells to grow
• Cells are fixed by gently mixing suspension of cells with 6 ml fixative . It is very
important to avoid clumping of the lymphocytes, the tubes containing cells
suspension in fixative and hypotonic solution are centrifuged at approximately
300 x g for 10mins
• The supernatant is removed and discarded into a labelled container for disposal
• The cells are resuspended by slow addition of fresh, cold fixative, mixing using a
vortex mixer to avoid cell clumping
• The tubes containing the suspension are centrifuged, this process is repeated
until the cells pallets no longer contain traces of red blood cells
30-04-2023 10
11. SAMPLING, SLIDE PREPARATION AND STAINING
• Lymphocytes are usually kept at 1-10°C at least overnight to ensure adequate
fixation
• Cells suspension are centrifuged and resuspended in a minimal amount of freshly
prepared fixative , several drops of cell suspension are placed on cold microscope
• After the slides have dried the cells are stained for 5 mins in filtered 4% Giemsa in
pH 6.8 Buffer
• The slides are rinsed, dried and mounted with coverslips
30-04-2023 11
12. STAINING
• Stain the slides in a solution of Hoechst 33258 for 25 mins protected from
sunlight
• Rinse slides thoroughly in MacIlvaine's buffer pH 8.0 twice, the second time
being in the buffer prewarmed to 40°C , place the slides flat in a tray and add
sufficient prewarmed buffer to cover the slides causing the chromatid arm to
swell
• Expose the slides to the UV light at approximately 366 nm for 25-40 mins
• Remove the slides and rinse thoroughly in PBS at pH 6.8
• Stain for 10 mins in filtered 4% Giemsa stain in PBS at pH 6.8, rinse the slides
thoroughly, first in PBS at pH6.8 and then in water, shake off excess moisture, air
dry and mount with coverslip
30-04-2023 12
13. ANALYSIS OF CELLS IN FIRST, SECOND AND THIRD
DIVISION
• The AGT is calculated as follows:
proliferative index(PI)= %cells M1 2(%cellsM2) 3(%cells M3)
100
Average Generation time= Hours in BrdU
PI
The AGT should be in the region of 12-14hrs for human lymphocytes, longer than
16hrs indicates that culture conditions are suboptimal and the source of the
problem should be identified and rectified
30-04-2023 13
14. ASSESSMENT OF MITOTIC INHIBITION
• Mitotic index = number of cells in mitosis X 100
Total no of cells observed
Mitotic inhibition%=[1-{mean Mlt/Mean Mlc}] X 100
Where Mlt= MI in cell exposed to test substance
Mlc= MI in solvent control cells
30-04-2023 14
15. ANALYSIS AND INTERPRETATION OF RESULTS
• After completion of analysis and decoding, the proportions of aberrant cells per
culture (often expressed as a percentage, since 100 cells are routinely analyzed)
are tabulated as:
• 1.Cells with structural aberrations including gaps
• 2.Cells with structural aberrations excluding gaps (this information is used to
draw a conclusion as to the clastogenic potential of a test substance as gaps may
occur by non-genotoxic modes of action)
• 3.Polyploid, endoreduplicated, or hyper diploid cells (An increase in polyploidy
may indicate that a chemical has the potential to induce numerical aberrations,
when use of the in vitro micronucleus test should be considered.)
30-04-2023 15
16. The acceptance and evaluation criteria to determine the outcome of the test
should be specified in advance. An acceptable test, assuming good cell growth,
would be likely to have:
1.Homogeneity between replicate cultures
2.The proportion of cells with structural aberrations (excluding gaps) in vehicle
control cultures should fall within the historical negative control range. In the
absence of an historical negative control range, an approximate guide would be
that the frequency of cells with aberrations should be <5%. This low frequency
means that there may not be any aberrations seen in 50 or even 100 cells.
3.At least 160 cells out of an intended 200 should be suitable for analysis at each
concentration of CPA, unless 10 or more cells per slide showing structural
aberrations other than gaps only have been observed during analysis.
30-04-2023 16
17. The following evaluation criteria may be used for ascribing the potential of a test
article to induce chromosome aberrations in a valid assay.
• A proportion of cells with structural aberrations at one or more concentrations
exceeds the concurrent vehicle control values and the historical negative control
(normal) range (if available) in both replicate cultures.
• A statistically significant increase in the proportion of cells with structural
aberrations (excluding gaps) is observed (p≤0.05).
• Evidence of a concentration-related trend in the proportion of cells with
structural aberrations (excluding gaps) will be judged to support the conclusion
• Results that only partially satisfy the above criteria need to be dealt with on a
case-by-case basis..
30-04-2023 17
18. • A test article that satisfies none of the above criteria may be considered not to
have the potential to induce chromosome aberrations. The criteria to establish a
negative response are more stringent, and two independent experiments are
required currently.
• The distinction between an indirect-acting and a direct-acting genotoxin is
important in predicting in vivo risk arising from a positive in vitro assay
30-04-2023 18