This document describes various in vitro models and techniques used to study hepatotoxicity, including hepatocyte cell cultures, viability assays, staining techniques, and molecular analysis methods. Specifically, it discusses using HepG2 and HuH7 cell lines to study hepatotoxicity. It also outlines methods like the Trypan Blue exclusion test, Oil Red O staining, MTT assay, RT-PCR, and Western blotting. Finally, it mentions using fatty acids like palmitic and oleic acids in in vitro models of non-alcoholic fatty liver disease.
German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death. Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis.
VIROMER® ONE RED - a standardized transfection reagent for plasmid DNA and mRNASandra Lagauzère
Viromer® ONE RED is a preformed and calibrated transfection reagent designed for in vitro delivery of plasmid DNA and mRNA. It comes in single portions of lyophilized material that enable standardized reactions and keep the product fresh until use. Reference data show high-performance transfection over 12 commonly used cell types including cancer and immune cells.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death. Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis.
VIROMER® ONE RED - a standardized transfection reagent for plasmid DNA and mRNASandra Lagauzère
Viromer® ONE RED is a preformed and calibrated transfection reagent designed for in vitro delivery of plasmid DNA and mRNA. It comes in single portions of lyophilized material that enable standardized reactions and keep the product fresh until use. Reference data show high-performance transfection over 12 commonly used cell types including cancer and immune cells.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
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
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
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
1. In vitro models of hepatotoxicity
Authors: Tea Omanović, Vjera Ninčević and Martina Smolić
Affiliation: Department of Pharmacology, Faculty of Medicine Osijek, University of Osijek,
J. Huttlera 4, 31000 Osijek, Croatia
1. Materials and methods used to study hepatotoxicity in vitro
1.1. Hepatocyte cell cultures
Two cell lines with different characteristics will be used for in vitro models of hepatotoxicity:
1. HepG2 cells are derived from a liver hepatocellular carcinoma of a 15 year old Caucasian
male and retain many characteristics of normal differentiated quiescent hepatocytes (1).
2. HuH7 cells are well differentiated hepatocytes derived from cellular carcinoma cell line
that was originally taken from a liver tumor in a 57-year-old Japanese male in 1982.
Above mentioned cell cultures are incubated with DMEM (Dulbecco's Modified Eagle Medium)
+ 10% FBS (fetal bovine serum) + 1% pen-strep (penicillin-streptomycin), in a humidified
incubator supplied with 5% CO2 at37 °C. Medium should be changed twice a week.
1.2. The Trypan Blue dye exclusion test
Cells viability in a suspension can be determined by the Trypan Blue dye exclusion test. Live cells
possess intact cell membranes that exclude certain dyes, among all trypan blue, whereas dead cells
do not (2). A cell suspension (if cells were cultured on plates, they should be detached prior to this
test) is simply mixed with the dye (eg. 10µl of cell suspension and 10µl of dye). Cells are counted
using hemocytometer (device that consists of a thick glass microscope slide with a rectangular
indentation that creates a chamber). Prior to using the hemocytometer, first make sure that the
special coverslip is properly positioned on the surface of the counting chamber. Apply cell
suspension to the edge of the coverslip to be sucked into the void by capillary action which
completely fills the chamber with the suspension. Afterwards, visually examine to determine if
cells take up or exclude dye. Clear cytoplasm will be present in viable cells whereas dead cells
will have a blue cytoplasm. Hemocytometer should be placed on the stage of a light microscope
and focus should be onto the cells. There are different methods of counting the cells, one of them
is to count just the viable cells with clear cytoplasm and then use this equation:
2. 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
𝑚𝑙
= 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
× 10 000
𝑐𝑒𝑙𝑙𝑠
𝑚𝑙
1.3. Oil red O staining
Oil red O is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and
lipids in different tissue samples. It has the appearance of a red powder with maximum absorption
at 518 (359) nm. 4% paraformaldehyde (cooled at 4°C) is used for cells fixation. After fixing the
cells for 30 - 45 min at 4 °C, remove fixative and rinse twice with cooled phosphate buffered saline
(PBS). Remove PBS and allow to air dry. Meantime, mix Oil red O stock (0.5% Oil Red O stock
consists of 0,5g Oil Red O diluted in 100ml of 99% isopropanol) at 6:4 ratio with distilled H2O
(dH2O) and let stand for 10 min. The working solution is stable for no longer than 2 hours, so
make up only what will be used in that time. Use a 0.2 micron syringe filter to add Oil red O to
cells. If the Oil Red O is not filtered properly you will have a lot of background staining. Slowly
rotate the dish to spread Oil Red O evenly over the cells. Leave the Oil red O for 10 min then
remove and rinse well with dH2O until the water runs clear. Add hematoxylin counterstain into
the well so that the cells are completely covered and let stand for 1 minute. Then remove and rinse
well with dH2O until the water runs clear. Leave the final dH2O rinse on the cells for microscopy
and analysis. Plates should be viewed on a phase contrast microscope where lipids will appear red
and the nuclei will appear blue.
1.4. MTT
The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay is a colorimetric
assay for evaluating cell metabolic activity and proliferation. Conversely, when metabolic events
lead to apoptosis or necrosis, the reduction in cell viability can be determined by MTT. The yellow
tetrazolium MTT is reduced by metabolically active cells, in part by the action of dehydrogenase
enzymes, to generate reducing equivalents such as NADH and NADPH (3). The resulting
intracellular purple formazan can be solubilized and the absorbance of this colored solution can be
quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a
spectrophotometer (3). MTT is suitable for investigation of drug hepatotoxicity, considering that
drug hepatotoxicity investigation often requires testing several different concentrations and drug
exposure times using cells in culture (4). Accordingly, it is attractive to use a viability test that
allows the analysis of many samples with little handling time, such as MTT (4). MTT assays should
3. be done in the dark since the MTT reagent is sensitive to light. First step is to plate cells, usually
in 96-well plate (e.g. 2x103 cells per well for a week long experiment) and incubate for 6-24 hours.
Next day treat cells with agents for appropriate time. Afterwards, remove medium and add fresh
medium with 0.5 mg/ml MTT. Incubate for 4-8 hours (depending of cell line, cell density etc.; e.g.
6 h for Huh7, Huh7.5 and 3T3 cells), until purple precipitate is clearly visible Remove MTT media.
Add equal volume of 0.04 M HCl in isopropanol or 10% SDS solution to solubilize cells. (3).
Read absorbance at 595 nm on Microplate reader. Absorbance values that are lower comparing to
the control cells signify a reduction in the rate of cell proliferation, as opposed to higher absorbance
rates which indicate an increase in cell proliferation (3).
1.5. RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR), represents variant of PCR preceded
with conversion of sample RNA into cDNA. This technique is frequently used in molecular
biology to detect gene expression through creation of cDNA transcripts from RNA using enzyme
reverse transcriptase. (5). Afterwards, amplification of the newly synthesized cDNA is done by
traditional PCR. First step of this method represents reverse transcription. Template RNA (1-10
µg), primers (1µl random oligos) and DEPC-H2O (DNase-RNase- free water) up to 14µl should
be mixed together in PCR tube, incubated at 70°C for 10 minutes, and cooled on ice. Afterwards,
add 7.8-8µl of reaction mix (2µl 10xPCR buffer, 1µl 50mM MgCl2, 1µl 10mM dNTPs, 2µl 0.1M
DTT (dithiothreitol), 1µl DEPC-H2O and 0.5-1µl reverse transcriptase) to each sample and
incubate 50 minutes at 42°C. Stop reaction by heating to 70°C for 15 minutes and cool on ice.
Subsequently, add 1µl RNase inhibitor (10µg) to each sample and incubate 20 minutes at 37°C.
At this moment, cDNA samples can be used for standard PCR (second step), or they can be stored
at 20°C for longer period of time. Second step begins by adding primers (10µl each) to 0.5 PCR
(0.5ml) tune. Create master mix of reagents, which consists of (for 100µl reaction): 10xPCR buffer
(10µl), 10mM dNTPs (2µl), 50mM MgCl2 (3µl) and Taq polymerase (0.5µl). Add 15.5µl of master
mix to each sample. Subsequently, add 1-10mg dilution of RT-PCR cDNA and DEPC-H2O up to
65µl. Overlay each sample with 3 drops of mineral oil. Finally, run samples (should be 100µl total)
in PCR cycle.
1.6. Western blotting
Western blotting is a widely used technique which enables detection of specific proteins in a
complex mixture of proteins extracted from cells (6). This technique can be divided into three
major steps: separation of proteins by size (gel electrophoresis), electrotransfer to a solid support
and finally blocking and antibody incubation (6). First step includes standard sample and gel
4. preparation preceding SDS-PAGE electrophoresis. Second step represents electrotransfer, which
makes proteins accessible to antibody detection by moving them from within the gel onto a
membrane made of nitrocellulose or polyvinylidene difluoride (PVDF) membrane (6).
Electroblotting uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose
membrane (proteins are negatively charged, therefore be sure to place membrane between the gel
and anode). Filter sheets and membrane should be cut to fit the measurement of the gel. Wet the
membrane in MetOH and the sponge and filter paper in transfer buffer. Afterwards create a transfer
sandwich which includes gel, membrane, a fiber pad (sponge) at each end, and filter papers to
protect the gel and blotting membrane. Move the sandwich to transfer apparatus and add transfer
buffer to the apparatus. Then place the electrodes and transfer for 45-90 minutes (depending on
the thickness of the gel). Last step begins by blocking the membrane (prevents non-specific
background binding of the antibodies) with 5% skim milk in TBST (Tris-Buffered Salin Tween-
20) for 1 hour. Afterwards, add primary antibody in 5% BSA and incubate (the incubation time
can vary between a few hours and overnight, depending on the binding affinity of the antibody)
on shaker. Wash the membrane 3 times with TBST for 5 minutes and then incubate with the
recommended dilution of conjugated secondary antibody in blocking buffer at room temperature
for 1 -2 hours. Agitation of the antibody is recommended to enable adequate homogenous covering
of the membrane and prevent uneven binding. Repeat the washing procedure. The secondary
antibody is usually labeled to a reporter enzyme such as horseradish peroxidase (HRP). The
membrane is therefore detected by the signal that labeled antibody (HRP plus enzyme substrate)
produces corresponding to the target protein position. Finally, by developing a film in a dark room,
signal is captured.
2. Fatty acids- in vitro models of NAFLD
Fatty acids (FFA), more precisely, palmitic (PA) and oleic acid (OA) represent determinants of
the pathophysiology of NAFLD (1). Palmitic and oleic acid are the most widely distributed
saturated and monounsaturated fatty acids in nature. Various studies have demonstrated that PA
and OA mixtures-induced steatosis is associated with lipotoxicity, liver injury, apoptosis and
steatosis in hepatocyte cell cultures (7). The amount of fat accumulation due to fatty acids activity
is comparable to that observed in livers of patients with NAFLD (8). Therefore, it is important to
master the knowledge and skills needed to develop in vitro models of NAFLD using free fatty
acids for a better understanding of disease itself.
5. 2.1. Fatty acids
Long-chain FAs, palmitic (16:0) and oleic (18:1) are provided as sodium salts. PA and OA are
dissolved in MetOH 99% (stock solution 100 mM). Stock solutions are kept at -20°C before the
experiments.
2.2. Protocol of the study—induction and evaluation of steatosis
HepG2 and HuH-7 cell cultures are incubated with above mentioned medium supplemented with
FA at the following final concentrations: a) PA: 0.33 mM and 0.66 mM; b) OA: 0.66 mM and 1.32
mM (1). Incubate control cell cultures with plain medium and with medium added with the vehicle
in which fatty acids were dissolved (in this case MetOH 99%). Figure 2.
Figure 2. 6-well plate with cell culture tested with FFAs as mentioned in text
1- Plain medium
2- Medium plus MetOH 99%
3- Medium plus 0.33mM PA solution
4- Medium plus 0.66mM PA solution
5- Medium plus 0.66mM OA solution
6- Medium plus 1.32mM OA solution
Various studies showed that optimal incubation period for getting the expected outcomes is 24h
hours (when incubating for 12h or less the triglyceride accumulation is low, following undetectable
variations in gene expression, in contrast, longer incubation period doesn’t provide a significant
advantage in terms of intracellular triglyceride accumulation, but significantly decreases cell
viability at higher FA concentrations) (1). Next step includes measurement of the extent of
steatosis, apoptosis and gene expression. Steatosis is measured by previously explained method
Oil-Red O staining (1.3), where lipid droplets should be colored in red, whereas nuclei in blue.
Level of apoptosis is determined by MTT (1.4). PA generally has higher ability to induce apoptosis
in cell cultures, compared to OA. Various studies use rabbit Phospho-Akt Antibody and Anti-β-
Actin antibody as primary antibodies for Western blot analyses. Finally, RT-PCR is used for
1 2 3
4 5 6
6. demonstrating gene expression. Expression of three genes has been found constantly changed after
treating cells with FAs: PPARα, PPARγ and SREBP-1 (1).
3. Acetaminophen induced hepatotoxicity
Acetaminophen (APAP) represents the most often used analgesic and antipyretic drug in general.
Nevertheless, hepatotoxicity caused by APAP overdose is the most common cause of acute liver
failure worldwide (9). To investigate all the molecular mechanisms involved in APAP
hepatotoxicity, various methods have to be used. Different studies offer in vitro approach to this
subject as one of the most effective ones. Primarily, incubate hepatic cell cultures in medium
containing APAP (0.5, 10, or 20 mM dissolved in 100% of dimethyl sulfoxide) for 2, 6, and 24 h
(10). To investigate the viability of hepatocytes through the mitochondrial function, the MTT assay
is used as previously described (9). Pparα, Faah, Nape-pld Mcp1, Tnfα, and Il6 genes represent
targets for measurement of gene expression by RT-PCR (10). Antibodies recommended for use
for protein expression by western blotting are PPARα, NAPE-PLD and FAAH. APAP which was
acutely and repeatedly administered to induce liver injury, modifies the expressions of the NAE-
based anti-inflammatory system composed by the PPARα, the NAEs synthesis enzyme NAPE-
PLD and the NAEs degradation enzyme FAAH. Significant decrease in expression of above
mentioned proteins was demonstrated 6h after treatment with APAP (10) . Interestingly, this effect
on proteins expression was completely reversed after 24 h from acute APAP administration (10).
4. Amiodarone induced hepatotoxicity
Amiodarone represents a class III antiarrhythmic medication used to treat and prevent various
types of arrhythmias. Amiodarone can have serious side effects, including hepatotoxic effect. More
precisely this drug can cause DIFLD (drug-induced fatty liver disease). Therefore, application of
amiodarone is mainly recommended only for serious ventricular arrhythmias. Better understanding
of amiodarone induced liver injury molecular mechanism is necessary. One of the ways of reaching
that goal is to assess the effect of amiodarone in hepatic cell cultures. First step includes incubation
of hepatic cell cultures with different concentrations of amiodarone (e.g. 10 µM and 20 µM of
amiodarone hydrochloride 98%, diluted in 99% MetOH) for 24h and 48h. Afterwards, different
tests can be done. The Trypan blue dye exclusion test can be performed to determine concentration
of viable cells. Oil-Red-O stain is used for demonstrating triglyceride accumulation in the
hepatocytes. Following incubation with amiodarone, cells change their normal shape to oval, larger
shape, with increased accumulation and size of lipid droplets into the cytosol of amiodarone-
7. treated cells compared to controls. For determining the exact toxic concentrations of amiodarone
and their effect on cell viability, MTT assay can be used. Western blot can be performed by using
antibodies against adipophilin and PPARγ.
5. Tamoxifen induced hepatotoxicity
Tamoxifen is a selective oestrogen receptor modulator commonly used in therapy of breast cancer,
particularly in molecular subtypes that express oestrogen receptors (11). Nevertheless, long-term
use of tamoxifen is associated with various complications, including drug induced fatty liver
disease. Accordingly, prevention and treatment of DIFLD in breast cancer patients require the
elucidation of the molecular mechanism of tamoxifen-induced DIFLD. Studies on hepatic cell
lines can play major role in elucidating this pathological mechanism. Same as for amiodarone, first
step includes incubation of hepatic cell cultures with different concentrations of tamoxifen (e.g. 5
µM and 10 µM of tamoxifen hydrochloride 98%, diluted in 99% MetOH) for 24h and 48h.
Afterwards, different tests can be done. The Trypan blue dye exclusion test can be performed to
determine concentration of viable cells. Tamoxifen decreases cells viability stronger then
amiodarone. Following Oil-Red-O staining of cells incubated with tamoxifen, change of their
normal shape to oval, larger shape, macrovesicular steatosis and phospholipidosis is visualized.
For determining the exact toxic concentrations of amiodarone and their effect on cell viability,
MTT assay can be used. When performing RT-PCR, expression of SREBP-1c, PPARγ and
C/EBPα genes has been changed (through various studies). Western blot analyses should include
antibodies for some of these proteins: SREBP-1c, FAS, C/EBPα, PPARγ and MTP (12).
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9. Jannuzzi AT, Kara M, Alpertunga B. Celastrol ameliorates acetaminophen-induced oxidative
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10. Rivera P, Pastor A, Arrabal S, Decara J, Vargas A, Sánchez-Marín L, et al. Acetaminophen-Induced
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two sides of the same coin. Eur Rev Med Pharmacol Sci. 2017;21(1 Suppl):86-94.
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hepatocyte steatosis in vitro. Int J Mol Sci. 2014;15(3):4019-30.