Selected Topic 2 nd Course - PowerPoint Presentation

Slide1

Selected Topic

2

nd

Course

Msc

. level

College of Pharmacy

Al-

Mustansiriyah

University

Dr.

Basma

Al-

Sudani

Lecture

4

27-5-2021

What Are the Indicators of Cell Viability?

Cell viability is a measure of the proportion of live, healthy cells within a population. Cell viability assays are used to determine the overall health of cells, optimize culture or experimental conditions, and to measure cell survival following treatment with compounds, such as during a drug screen.

Typically, cell viability assays provide a readout of cell health

through measurement of metabolic activity, ATP content, or

cell proliferation. Cell viability can also be assessed using cell

toxicity assays that provide a readout on markers of cell death

such as a loss of membrane integrity.

Together, cell viability and cell toxicity assays are important tools for assessing cellular responses to experimental compounds of

interest.

Role of Cell Proliferation as an Indicator of Cell

Viability

Cell proliferation assays can measure cell division events directly (counting cells) or use markers of cell division/DNA replication such as DNA content.

Cell proliferation refers to an increase in cell number due to cell division (cytokinesis), which occurs as the final step of the cell cycle. Healthy cells actively proliferate whereas growth-arrested, senescent, and dead or dying cells do not. Thus, cell proliferation assays are a useful tool for assessing cell viability or cell survival by providing a readout on the number of actively dividing cells present in a sample.

How to Measure Cell

Proliferation

Cell proliferation assays typically measure DNA content or DNA synthesis in replicating cells. Cell proliferation assays are performed using standard methods, including enzyme-linked

immunosorbent

assay (ELISA), flow

cytometry, immunofluorescence and high content imaging.

Cell cycle assay—analysis of DNA content

Cell cycle assays are used to determine the proportion of cells at different stages of the cell cycle via flow

cytometry

. During cell cycle progression, cells increase in size (G

1

 phase), which is followed by DNA synthesis and replication (S phase), further growth (G

2 phase), and finally by mitosis (M phase) and cell division. Cells that exit the cell cycle and stop dividing remain at resting state (G0

) and are referred to as quiescent.

Phases of the Cell Cycle. G1 - increasing size; S - DNA synthesis and replication; G2 - growth; M - mitosis and cell division; G0 - quiescent.

For the cell cycle assay, cells are fixed or permeabilized

, incubated with a DNA intercalating agent such as propidium iodide (PI) and subsequently analyzed by flow cytometry

. Differences in fluorescence intensity of the PI signal correlate to different phases of the cell cycle. For example, cells in G2 or M phase have twice as much DNA content as cells in G

0 or G1 phase, which corresponds to an increased fluorescence signal. Cells in S phase have varying fluorescence intensities which are between that observed for cells in G

0/G

1 and G2/M phase. Fluorescence signals below the G

0/G1 peak typically correspond to apoptotic cells with fragmented DNA.

Proliferation assay—analysis of DNA synthesis

During S phase, nucleoside labeling agents such as 3H-thymidine or 5-bromo-2'-deoxyuridine are incorporated into newly synthesized DNA. The level of reagent incorporation is proportional to the amount of cell division in the target population. While the 3H-thymidine incorporation assay requires the use of radioactivity,

BrdU

incorporation is detected using an 

anti-

BrdU

antibody in ELISA, flow cytometry

, or IF-IC applications and does not require the use of radioisotope labeling.

C2C12 cells were seeded at varying density in serum free medium in a 96-well plate and incubated overnight. Serum was added to the plate at various concentrations and cells were incubated for 24 hr. Finally, 10

μM

BrdU was added to the plate and cells were incubated for 4

hr

Detection of proliferation proteins

Dividing cells have high expression of cell cycle proteins compared to quiescent or senescent cells, thus the level of cell cycle-specific proteins can be measured as a readout of cell proliferation. Some common proliferation proteins include 

proliferating cell nuclear antigen (PCNA)

, 

Ki67

, and 

Phospho-histone H3, which can be detected using western blot (WB), IF, IHC, flow

cytometry, and ELISA.

Immunohistochemical

analysis of paraffin-embedded human breast carcinoma using Ki-67 (8D5) Mouse mAb

.

Senescence to indicate impaired proliferation

Dysfunctional replication can result in senescence, and senescent cells will not divide in response to growth signals. Markers of senescence can therefore be used as a complement to cell proliferation assays. An enlarged cell size, expression of pH-dependent 

β-

galactosidase

activity

, and an altered pattern of gene expression further characterize senescent cells and assist assessment of cell proliferation.

β-

Galactosidase

staining at pH 6.0 on MCF-7 cells untreated (left) and senescent MCF-7 cells treated with etoposide #2200 (12.5

μM, 24 hr) and allowed to recover for 4 days (right).

Cell Viability

Cell viability assays are used to determine the number of healthy cells in a sample. Aside from proliferation assays described above, other cell viability assays report on the overall health of a population, without distinguishing dividing cells from non-dividing cells.

How to Test Cell Viability

The most common readout of cell viability is with vital dyes such as 

propidium

iodide

, however cell viability assays also typically measure the metabolic activity or ATP content of healthy cells.

Metabolic assays such as the MTT and 

XTT assays

 quantify cell health by measuring reduction of a colorimetric substrate by mitochondrial enzymes. The MTT assay quantifies the relative quantity of viable cells using this approach. Cultures are incubated with the yellow

tetrazolium dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) which, in healthy cells, is converted by mitochondrial enzymes into an insoluble purple formazan

product. After solubilization by detergents or isopropanol, the number of viable cells can be determined by measuring absorbance at 450 nm in a

microplate reader. The XTT cell viability assay is an alternative to the MTT assay which yields a formazan product that is soluble in aqueous solutions, and thus does not require an additional

solubilization step.

C2C12 cells were seeded at varying density in a 96-well plate and incubated overnight. The XTT assay solution was added to the plate and cells were incubated. The absorbance at 450 nm was measured at 1.0, 2.0, 3.0, 4.0, and 5.0 hours.

1- Seeded the cells in 96 well plate

Protocols of

MTT, MTS, XTT

2- Treated the cells with compounds

Analyze the results

ATP measurement assays quantify ATP content to determine the number of viable, metabolically active cells in a sample. These assays are performed in

multiwell

plates with a colorimetric,

fluorometric

, or luminescent readout of a metabolic activity requiring ATP, where substrate generation is proportional to the number of healthy cells with active mitochondria.

ATP measurement

Cell Toxicity

Cell toxicity refers to the ability of a substance to damage or kill cells. Cell toxicity can be assessed directly by assays that quantify markers of cell death, such as a loss of membrane integrity. Cell toxicity can also be assessed indirectly using the cell viability assays.

Cell toxicity assays are useful tools for assessing cell survival following treatment with pharmacological agents or other stressors as well as examining the effect of compounds on immune cell mediated killing of tumor cells using the T cell killing assay.

What is Cell Toxicity?

Cell toxicity refers to the ability of a substance to damage or kill cells.

How to Test Cell Toxicity

Many cell toxicity (cytotoxicity) assays are available that report on different indicators of cell death or compromised cell health.

Live/dead cell counting

 is used to measure the quantity of both live and dead cells in the sample population. For example, the

trypan

blue dye exclusion method relies on the principle that only dead or dying cells with damaged membranes take up

trypan

blue so the number of dead (blue) or viable (colorless) cells can be counted using a hemocytometer.

The Cell Health Assay

 is a fluorescence-based assay used for live/dead cell counting that is amenable to fluorescence microscopy and flow

cytometry

analyses wherein live cells are detected by the conversion of

calcein-AM to

calcein and dead cells by uptake of propidium iodide. Live-cell imaging platforms can also be used to monitor cell death in real time

.Cell membrane integrity is assessed to determine if cells have damaged or ruptured membranes due to primary necrosis or secondary necrosis following apoptosis. Membrane damage causes release of cytosolic contents into the extracellular space including the enzyme lactate dehydrogenase (LDH). The amount of extracellular LDH can be analyzed using a colorimetric assay where the amount of product formed correlates to the quantity of dead or damaged cells in the sample. Cell membrane integrity can also be assessed by analyzing the uptake of membrane impermeable dyes such as

propidium

iodide.

T cell killing assays

 are used to examine the effect of test compounds on immune cell-mediated killing of cultured tumor cells. Cytotoxic T cells are a class of T cells that recognize and destroy virus-infected cells and tumor cells. In the T cell killing assay, target tumor cells are first labeled with a reporter that permits identification of live versus dead cells. Test compound and cytotoxic T cells are then added to the culture and tumor cell viability monitored using live cell imaging or flow

cytometry

.

Treatment of

HeLa

cells (4 x104 cells/well) with increasing concentrations of

terfenadine

(4 hr) results in reduced cell viability as detected by the Cell Health Assay Kit.

Apoptosis

Apoptosis is a form of programmed cell death necessary for proper growth, development, and homeostasis of multicellular organisms, but can also occur in response to cellular stress. This is different than cell death by necrosis,

necroptosis

, and

pyroptosis

. During apoptosis cells undergo characteristic shrinkage, membrane

blebbing, nuclear fragmentation, and chromatin condensation. Cell debris is packaged into apoptotic bodies which are then engulfed and digested by phagocytes.

Apoptosis is mediated by a class of

proteolytic enzymes called

caspases which can be activated via intrinsic (loss of mitochondrial membrane potential and cytochrome c release) or extrinsic (ligand binding to death receptors) apoptotic signaling mechanisms.

Why Does Cell Apoptosis Occur?

Apoptosis occurs normally during growth and development to eliminate cells that are no longer needed or that are potentially harmful to the organism, such as virus-infected cells or cells harboring damaged DNA. Dysregulation of apoptosis occurs in disease, where elevated levels of apoptosis are associated with autoimmune and neurodegenerative disorders and reduced levels of apoptosis occur in many cancers.

Apoptosis can be induced experimentally by incubating cells with ligands that bind 

death receptors

 including TNF-R1 and TNF-R2 or with pro-apoptotic chemical agents like

staurosporine

or

etoposide.

How to Measure Apoptosis

Apoptosis assays

 are used to quantify the number of cells undergoing apoptosis. Examples include detection of

annexin

V binding to exposed membrane lipids,

caspase activation, chromatin condensation, DNA fragmentation, or cytochrome c release.

Annexin

V assay membrane asymmetry, or the irregular distribution of different lipid species on either side of the plasma membrane, is disrupted during apoptosis as phosphatidylserine

(PS) translocates from the inner to the outer leaflet of the plasma membrane. The presence of PS on the outer leaflet signals for engulfment and degradation by phagocytes. Exposed PS can be recognized by

fluorophore-conjugated annexin V

 using flow cytometry, IF, or ELISA as an early marker of apoptosis.

Flow

cytometric

analysis of Jurkat

cells untreated (left) or treated with camptothecin (10μM, 4 hr; right) using

Annexin V-FITC Early Apoptosis Detection Kit.

Caspase assay

c

aspase

enzymes are processed from inactive zymogens to active proteases during the initiation and execution of apoptosis. The initiator caspases-8 and -9 activate executioner

caspases

like caspase-3. Caspase-3 cleavage

 can be detected via western blotting, IF, IHC, or flow cytometry as a readout for apoptosis. Caspase-3 activity

 and caspase substrates like cleaved PARP can also be measured using methods similar to cleaved caspase-3 or by ELISA where the extent of substrate processing is proportional to the quantity of apoptotic cells in the sample. Further examination of apoptotic pathways can be performed using antibodies to 

other activated caspases.

Caption Western blot analysis of extracts from

HeLa

, NIH/3T3 and C6 cells untreated, staurosporine-treated (3hrs, 1 µM in vivo) or cytochrome c-treated (1hr, 0.25 mg/ml in vitro), using Caspase-3 Antibody (upper) or Cleaved Caspase-3 (Asp175) Antibody (lower).

TUNEL assay

DNA strand breaks and DNA fragmentation are characteristic of late-stage apoptosis and can be detected using terminal

deoxynucleotidyl

transferase (TdT

) dUTP nick end labeling (TUNEL assay) to quantify the number of apoptotic cells within a population via IF, IHC, plate reader, or flow cytometry

. The TdT enzyme catalyzes addition of labeled

dUTPs, such as dUTPs conjugated with a

fluorophore or biotin, at sites of DNA double strand breaks. Apoptotic cells or other cells with DNA damage are labeled with the modified dUTP while healthy cells are not. Fragmented DNA from apoptotic cells can also be observed using standard gel electrophoresis, as the fragmented DNA will appear laddered compared to the intact DNA from healthy cells.