flow cytometry-based quantification of therapy-induced senescent cancer cells

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

First, adjust the lysosomal pH of cultured cell samples by adding a solution of one micromolar bafilomycin A in DMEM to the cell pellet at a concentration of one times 10 to the sixth cells per milliliter, and pipette to mix. Incubate for 30 minutes at 37 degrees Celsius on a rotator at a slow speed. Then, to stain for senescence-associated beta-galactosidase, add DDAOG stock solution at 10 micrograms per milliliter to the samples without washing.
Place on a rotator for 60 minutes, protected from direct light. As before, centrifuge the tubes. Remove the supernatant, and wash the pellet with one milliliter of ice-cold 0.5% BSA in PBS. Then, add 300 microliters of diluted calcine violet 450 AM solution to the washed cell pellets. Incubate for 15 minutes on ice in the dark.
Transfer the cell samples to flow cytometry instrument-compatible tubes. In the data acquisition software, open a violet channel histogram and a far-red channel versus green channel dot dot-plot. Initiate cytometer data acquisition at a low intake speed and place the positive control samples stained with DDAOG on the intake port. Begin to acquire sample data. Adjust the channel voltages such that more than 90% of events are contained within each plot.
When all settings are optimal, record 10,000 events per sample. Place the vehicle-only control samples stained with DDAOG on the intake port. Initiate data acquisition and record 10,000 events per sample. Look for an increase in autofluorescence or AF and DDAO galactoside signal versus positive control. Save the sample data in .FCS file format and export the files to a workstation computer equipped with flow cytometry analysis software.

flow-cytometry-based-quantification-therapy-induced-senescent-cancer

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

EoE Sub Articles

eoe sub articles

1. Induction of senescence by chemotherapy drugs in cultured cancer cells
NOTE: All cell manipulation steps in this section should be performed in a biosafety cabinet using sterile practices. This section is written for adherent cell types. Suspension cells may be used with appropriate modifications as noted.
<ol>
	<li>Grow cancer cell line(s) according to the standard protocol from the supplier or the laboratory that provided the specific cell line(s) used. 
	NOTE: Low passage cells (p &#60; 10) are generally preferred as there will be lower levels of replicative senescence, i.e. lower background, in untreated cell samples.</li>
	<li>One day prior to senescence induction by drugs, harvest cells with trypsin-EDTA 0.25% (or as recommended). Neutralize trypsin by adding an equal volume of complete culture medium, and transfer the cell suspension to a sterile conical tube. 
	NOTE: This step is not needed for suspension cells.</li>
	<li>Count the cells using the standard hemacytometer method and record cells/mL. Plate cells at 1 &#215; 103-10 &#215; 103 cells/cm2 in standard 6-well plastic culture dishes. 
	NOTE: Optimal plating density is dependent on the proliferation rate of cells and should be user-determined. Cells should be in log phase growth at approximately 10%-20% confluency at the time of treatment (i.e., following 18-24 h incubation after plating). The starting density (cells/mL) of suspension cells should be user-determined. Six-well plates typically yield enough senescent cells per well for one standard flow cytometry analysis sample. If flow-sorting, a much larger surface area (e.g., multiple P150 plates) should be used to enable the recovery of sufficient numbers of senescent cells for downstream assays (&#8805;1 &#215; 106).</li>
	<li>Incubate plated cells overnight (18-24 h) in a 37 &#176;C incubator with 5% CO2 and a humidity pan.</li>
	<li>Treat the plated cells with senescence-inducing chemotherapy drug(s). Include at least one positive control, e.g., etoposide (ETO) or bleomycin (BLM). Prepare duplicate wells per drug. Include one set treated with vehicle-only as control. 
	NOTE: A dose curve for each experimental agent should be tested by the user to determine the optimal concentration for senescence induction in the cell line(s) being used.</li>
	<li>Incubate cells for 4 days in a 37 &#176;C incubator with 5% CO2 and a humidity pan to allow the onset of senescence. Examine daily for expected morphology changes using a light microscope. 
	NOTE: Incubation times from 3-5 days may be acceptable depending on the rate of senescence onset. Media can be changed and the agent reapplied (or not), as desired, to promote healthy growth conditions while achieving an acceptable percentage of senescent cells.</li>
	<li>After the onset of senescence, harvest the cells by adding trypsin-EDTA 0.25% for 5 min at 37 &#176;C. When cells are dissociated into suspension, neutralize trypsin with an equal volume of complete medium. 
	NOTE: This step is not needed for cells growing in suspension. If surface marker staining will be conducted, avoid the use of trypsin-EDTA as it can temporarily destroy surface antigens on cells. Instead, gently dissociate the monolayer using a sterile plastic cell scraper (or an alternate dissociation reagent designed to preserve surface antigens).</li>
	<li>Count the cells in each sample using a hemacytometer. Calculate the cells/mL for each sample. 
	NOTE: Trypan blue can be added to evaluate the percentage of dead cells at this point (i.e., due to drug treatment), but cell death will also be determined with a fluorescent viability dye during DDAOG staining workflow.</li>
	<li>Aliquot &#8805;0.5 &#215; 106 cells per sample into 1.7 mL microcentrifuge tubes. 
	NOTE: The number of cells per sample should be standardized across all samples.</li>
	<li>Centrifuge the tubes for 5 min at 1,000 &#215; g in a microcentrifuge at 4 &#176;C. Remove the supernatant. 
	NOTE: If a refrigerated microcentrifuge is unavailable, it may be acceptable to perform centrifugations at ambient temperature for certain resilient cell types.</li>
	<li>Proceed to DDAOG staining in section 2.</li>
</ol>
2. DDAOG staining of SA-&#946;-Gal in cell samples
<ol>
	<li>Dilute 1 mM Bafilomycin A1 stock at 1:1,000x into DMEM medium (without FBS) for a final concentration of 1 &#181;M.</li>
	<li>Add prepared Baf-DMEM solution to the cell pellet samples (from step 1.11) for a concentration of 1 &#215; 106 cells/mL. 
	NOTE: For example, if using 0.5 &#215; 106 cells per sample, add 0.5 mL of Baf-DMEM.</li>
	<li>Incubate for 30 min at 37 &#176;C (without CO2) on a rotator/shaker set at a slow speed. 
	NOTE: Avoid CO2 incubators for the staining process, which can acidify solutions and thereby interfere with Baf and DDAOG staining.</li>
	<li>Without washing, add the DDAOG stock solution (5 mg/mL) at 1:500x (10 &#181;g/mL final) to each sample. Pipette to mix. Replace on a rotator/shaker at 37 &#176;C (without CO2) for 60 min. Protect from direct light.</li>
	<li>Centrifuge the tubes for 5 min at 1,000 x g at 4 &#176;C. Remove the supernatant.</li>
	<li>Wash with 1 mL of ice-cold 0.5% BSA per tube and pipette to mix. Centrifuge the tubes for 5 min at 1,000 x g at 4 &#176;C and remove the supernatant. Repeat this step 2x to thoroughly wash the cells. Remove the supernatant and proceed. 
	NOTE: It is important to perform the wash steps in step 2.6 to remove uncleaved DDAOG, which can exhibit undesired fluorescence emission (460/610 nm).</li>
	<li>(Optional) Fixation and storage of DDAOG stained cells for later analysis
	<ol>
		<li>Add 0.5 mL of ice-cold 4% paraformaldehyde dropwise to each washed sample. Pipette to mix.</li>
		<li>Incubate for 10 min at room temperature.</li>
		<li>Wash the cells 2x with 1 mL of PBS.</li>
		<li>Store the samples for up to 1 week at 4 &#176;C prior to flow cytometry analysis. 
		NOTE: For fixed samples, skip step 2.8.</li>
	</ol>
	</li>
	<li>Dilute Calcein Violet 450 AM stock (1 mM) at 1:1,000x into 1% BSA-PBS (1 &#181;M final). Add 300 &#181;L (for cultured cell samples) to the washed cell pellets from step 2.6. Incubate for 15 min on ice in the dark.</li>
	<li>Proceed to flow cytometry setup (section 3).</li>
</ol>
3. Flow cytometer setup and data acquisition
<ol>
	<li>Transfer the cell samples to tubes compatible with the flow cytometry instrument. Place the tubes on ice and keep them protected from light. 
	NOTE: If aggregates are observed in the cell suspensions, pass the suspension through 70-100 &#181;m cell strainers prior to analysis. Do not use 40 &#181;m strainers because they can exclude some of the larger senescent cells.</li>
	<li>In the referenced software (see the Table of Materials), open the following plots: 1) FSC-A vs SSC-A dot plot, 2) violet channel histogram, 3) far-red channel (e.g., APC-A) versus green channel (e.g., FITC-A) dot plot. 
	NOTE: Doublet exclusion plots and single-channel histograms can also be used but are not strictly required.</li>
	<li>Initiate cytometer data acquisition.
	<ol>
		<li>Place the vehicle-only control sample stained with DDAOG on the intake port. At a low intake speed, begin to acquire sample data.</li>
		<li>Adjust FSC and SSC voltages so that &#62;90% of events are contained within the plot. If cells do not fit well on the plot, lower the area scaling setting to 0.33-0.5 units.</li>
		<li>Remove the vehicle-only sample without recording data.</li>
		<li>(Optional) Add one droplet of rainbow calibration microspheres to a cytometer tube with 1 mL of PBS. Place the tube on the cytometer intake port. Begin to acquire sample data.</li>
		<li>Adjust violet, green, and far-red channel voltages so that the top peak of the rainbow microsphere is in the range of 104-105 units of relative fluorescence in each channel and all peaks are well separated in each channel. Record 10,000 events. Remove the tube.</li>
	</ol>
	</li>
	<li>Place the positive control sample (e.g., BLM, ETO) stained with DDAOG on the intake port. At a low speed, acquire sample data. Observe the events in FSC, SSC, violet, green, and far-red channels to ensure that over 90% of events are contained within all plots. Look for an increase in AF and DDAOG signal versus vehicle-only control.</li>
	<li>If using a sorting cytometer, initiate sorting at this step.
	<ol>
		<li>For record-keeping purposes, record 10,000 cells for the control sample and each sorted sample.</li>
		<li>Sort the desired amount of cells (&#8805;1 &#215; 106 is typically suitable) into an instrument-appropriate collection tube with 3-5 mL of culture medium.</li>
		<li>After sorting, proceed to downstream culture or analysis. 
		NOTE: For the calibrated flow cytometer used herein, optimal channel voltages typically fell between 250 and 600 (mid-range), but the optimal voltages and channel voltage ranges will vary across instruments. Avoid using voltages at very low or high ranges, which may suppress signal or amplify noise.</li>
	</ol>
	</li>
	<li>After completing steps 3.1-3.5 and making adjustments to the cytometer settings as necessary, record data for all the samples. Ensure that the settings remain uniform for all sample recordings. Record &#8805;10,000 events per cultured cell sample. 
	NOTE: Although gating and analysis can be performed using data acquisition software (e.g., FACSDiva), a complete gating and analysis workflow to be conducted post-acquisition using separate analysis software (FlowJo) is described in section 4 below. Post-acquisition analysis is preferred to reduce time at the cytometer workstation and take advantage of additional tools included in the dedicated analysis software.</li>
	<li>Save sample data in .fcs file format. Export the files to a workstation computer equipped with flow cytometry analysis software (e.g., FlowJo).</li>
</ol>
4. Flow cytometry data analysis
NOTE: The workflow presented uses FlowJo software. Alternative flow cytometry data analysis software may be used if the key steps described in this section are similarly followed.
<ol>
	<li>Using FlowJo software, open .fcs data files from step 3.7.</li>
	<li>Open the layout window.</li>
	<li>Drag and drop all samples into the layout window.</li>
	<li>Gate viable cells.
	<ol>
		<li>First double-click on the sample data for the vehicle-only control to open its data window.</li>
		<li>Visualize the data as a violet channel histogram. Identify the viable cells stained by CV450 based on their brighter fluorescence than the dead cells.</li>
		<li>Draw a gate using the single-gate histogram tool to include viable cells only. Name the gate viable.</li>
		<li>Then, from the sample layout window, drag the viable gate onto the other cell samples to apply the gate uniformly.</li>
		<li>In the layout window, visualize all samples as violet channel (viability) histograms. Verify that viable cell gating is appropriate across samples before proceeding; if not, make adjustments as needed. 
		NOTE: Viability staining can exhibit variations across treatments.</li>
	</ol>
	</li>
	<li>Gate senescent cells.
	<ol>
		<li>Double-click on the gated viable cell data for the vehicle-only control to open its data window.</li>
		<li>Visualize the data as a dot plot for far-red channel (DDAOG) vs green channel (AF).</li>
		<li>Draw a gate using the rectangle gating tool to include &#60;5% of cells that are DDAOG+ and AF+ (upper right quadrant). Name the gate senescent.</li>
		<li>Then, from the sample layout window, drag the senescent gate onto the viable subsets of the other cell samples to apply the gate uniformly.</li>
		<li>Into the layout window, drag and drop all viable cell subsets gated in section 4.4. Visualize all viable samples as far-red (e.g., APC-A) versus green channel (e.g., FITC-A) dot plots.</li>
		<li>Ensure that the senescent gate drawn in step 4.5.3 is visible on all plots and that the gate for the vehicle-only control exhibits &#8804;5%-10% senescent cells.</li>
	</ol>
	</li>
	<li>Once the percentage of senescent cells has been determined using the steps above, present the resulting data using the FlowJo plots, summarized in a data table, and/or statistically analyzed using standard software.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Research Products International</td>
			<td>B40500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin sulfate</td>
			<td>Cayman</td>
			<td>13877</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A1380</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein Violet 450 AM viability dye</td>
			<td>ThermoFisher Scientific</td>
			<td>65-0854-39</td>
			<td>eBioscience</td>
		</tr>
		<tr>
			<td>DPP4 antibody, PE conjugate</td>
			<td>Biolegend</td>
			<td>137803</td>
			<td>Clone H194-112</td>
		</tr>
		<tr>
			<td>Cell line: A549 human lung adenocarcinoma</td>
			<td>American Type Culture Collection</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell line: B16-F10 mouse melanoma</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers, 100 &#181;m</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>DDAO-Galactoside</td>
			<td>Life Technologies</td>
			<td>D6488</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium 1x</td>
			<td>Life Technologies</td>
			<td>11960-069</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, hydrochloride injection (USP)</td>
			<td>Pfizer</td>
			<td>NDC 0069-3032-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, PEGylated liposomal (USP)</td>
			<td>Sun Pharmaceutical</td>
			<td>NDC 47335-049-40</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M</td>
			<td>Life Technologies</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Cayman</td>
			<td>12092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc receptor blocking reagent</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>Anti-mouse CD16/32</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell analyzer)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>LSRFortessa</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell sorter)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>FACSAria</td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Lonza</td>
			<td>BW17737</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Sigma</td>
			<td>5401020001</td>
			<td>Roche</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Corning</td>
			<td>MT21031CV</td>
			<td>Dulbecco&#39;s PBS (without calcium and magnesium)</td>
		</tr>
		<tr>
			<td>Rainbow calibration particles, ultra kit</td>
			<td>SpheroTech</td>
			<td>UCRP-38-2K</td>
			<td>3.5-3.9 &#181;m, 2E6/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium 1x</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% (USP)</td>
			<td>Baxter Healthcare Corporation</td>
			<td>2B1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for cytometer data acquisition, &#34;FACSDiva&#34;</td>
			<td>Becton Dickinson (BD)</td>
			<td>n/a</td>
			<td>Contact BD for license</td>
		</tr>
		<tr>
			<td>Software for cytometer data analysis, &#34;FlowJo&#34;</td>
			<td>TreeStar</td>
			<td>n/a</td>
			<td>Contact TreeStar for license</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Life Technologies</td>
			<td>25200-114</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Flor, A., et al. <a href="https://app.jove.com/v/64176">Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.</a> J. Vis. Exp. (2022)
This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a &#946;-galactosidase substrate. The substrate is cleaved by active lysosomal &#946;-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

1. Induction of senescence by chemotherapy drugs in cultured cancer cells
NOTE: All cell manipulation steps in this section should be performed in a ...

Take cancer cells pre-treated with a chemotherapy drug that induces senescence in a percentage of cells.
These cells accumulate autofluorescent lipofuscin granules, a known senescence marker.
Add an antibiotic that enters the cells and inhibits the lysosomal proton pumps, preventing lysosomal acidification.
An increase in pH inactivates the lysosomal enzymes.
Senescent cells overexpress lysosomal &#946;-galactosidase even at near-neutral pH, making it a senescence biomarker.
Introduce a &#946;-galactosidase substrate. Incubate.
&#946;-galactosidase hydrolyzes the internalized substrate into a fluorescent product, labeling the senescent cells.
Centrifuge and discard the supernatant.
Add a cell viability dye. Within live cells, active esterases convert the dye into a membrane-impermeable fluorescent product. Dead cells with compromised membrane integrity remain unstained.
Using flow cytometry, detect the viable cells displaying the dual markers lipofuscin and fluorescent &#946;-galactosidase substrate, indicating senescent cells.
Quantify the percentage of senescent cells to demonstrate the drug efficacy.

21 Görüntüleme. Tedaviye Bağlı Yaşlanan Kanser Hücrelerinin Akış Sitometrisine Dayalı Miktar Tayini

Video: Tedaviye Bağlı Yaşlanan Kanser Hücrelerinin Akış Sitometrisine Dayalı Miktar Tayini - Deney

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. Here, we describe a quick and cost-effective one-step assay for simultaneously assessing the major modes of clonogenic cell death induced by IR, i.e., apoptosis, mitotic catastrophe, and cellular senescence. In this method, cells grown on a cover slip are irradiated with X-rays and stained with 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI). Using fluorescence microscopy, apoptosis, mitotic catastrophe, and cellular senescence are identified based on the characteristic morphologies of the DAPI-stained nuclei. Apoptosis is determined by the presence of apoptotic bodies (i.e., condensed and fragmented nuclei). Mitotic catastrophe is determined by the presence of nuclei that exhibit two or more distinct lobes and micronuclei. Cellular senescence is determined by the presence of senescence-associated heterochromatic foci (i.e., nuclear DNA containing 30-50 bright, dense foci). This approach allows the experimenter to easily screen for clonogenic cell death modes using various cell lines, treatment settings, and/or time points, with the goal of elucidating the mechanisms of cell death in the target cells and conditions of interest.

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Tek adımlı protokol modu Floresans mikroskobu tarafından radyasyon kaynaklı klonojenik progenitör mobilizasyonu yapar hücre ölümü değerlendirilmesi için

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effect of IR on malignant tumors and normal tissues. ...

JoVE Journal

Kanser Araştırmaları

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Tedaviye Bağlı Yaşlanan Kanser Hücrelerinde Çoklu Floresan Yaşlanma Belirteçlerinin Eşzamanlı Görüntüleme ve Akış Sitometrisine Dayalı Tespiti

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Metabolik Etiketleme ve Kütle Spektrometrisi ile Yaşlanan ve Bölünmeyen Kültürlü Hücrelerde Protein Devir Hızlarının Ölçülmesi

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

TRANSKRIPT

Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells