This work was supported by a grant to Yong Yu from Johannes Kepler University Linz (BERM16108001).

1. DLBCL cell lines with mafosfamide or daunorubicin treatment to induce cellular senescence
NOTE: The protocol also works for adherent cancer cells. Depending on cell size, seed 1-2 &#215; 105 cells into one well of a 6-well plate and incubate the plate in a 5% CO2, 37 &#176;C incubator overnight before treatment. The protocol steps are the same as for suspension cells but with two exceptions. First, cells need to be trypsinized off the plate after step 3.4. Second, wash steps are performed without centrifugation before trypsinization.
<ol>
	<li>Count DLBCL cells and seed 1 &#215; 106 cells/mL in 4 mL of medium per well into a 6-well culture plate. Cultivate DLBCL cells in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin.</li>
	<li>Add MAF (5 &#181;g/mL) or DN (20 ng/mL) into the cell culture and gently rock the plate to mix. 
	NOTE: MAF and DN are not stable. After dissolving in DMSO, the stock solution should be aliquoted and stored at -20 &#176;C. Freeze/thaw cycles should be avoided.</li>
	<li>Incubate the plate in a 5% CO2, 37 &#176;C incubator for 3 days.</li>
	<li>After a 3 day incubation, collect the DLBCL cells in 15 mL sterile centrifuge tubes and spin at 100 &#215; g, 4 &#176;C for 5 min.</li>
	<li>Discard the supernatant, resuspend the cell pellets with 4 mL of fresh medium, and add the suspensions back into the 6-well plate.</li>
	<li>Cultivate the cell plate in a 5% CO2, 37 &#176;C incubator for another 2 days before harvesting for analysis.</li>
</ol>
2. Prepare solutions for staining (Table 1)
3. Stain DLBCL cells with different senescence markers
NOTE: Cell samples stained with individual markers (i.e., pacific blue-EdU, C12FDG, or Alexa Fluor 647-&#947;H2AX) are prepared to generate a compensation matrix to correct the fluorescence spillover during measurement. Although highly suggested, this step could be suspended when there is an omittable overlap (compensation coefficient value &#8804; 0.1) of the emission spectra among different fluorophores. However, users must determine standardized compensation steps when using different instruments and fluorescent panels.
<ol>
	<li>Add 10 mM EdU solution at a ratio of 1:1,000 into the DLBCL cell culture generated from step 1.6 (final EdU concentration is 10 &#181;M). Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 3 h.</li>
	<li>Take the plate out of the incubator and add 100 mM chloroquine solution to a final concentration of 75 &#181;M (see discussion). Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 30 min.</li>
	<li>Take the plate out of the incubator and add 20 mM C12FDG solution at 1: 1,000 to a final C12FDG concentration of 20 &#181;M. Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 1 h.</li>
	<li>Take the plate out of the incubator and add 100 mM 2-phenylethyl-&#946;-D-thiogalactoside (PETG) solution at 1:50 to stop the fSA-&#946;-gal staining (final PETG concentration of 2 mM). Gently rotate the plate to mix.</li>
	<li>Transfer the cells to 15 mL sterile centrifuge tubes and spin at 100 &#215; g, 4 &#176;C for 5 min. Discard the supernatant and wash the cells with 4 mL of phosphate-buffered saline (PBS).</li>
	<li>Repeat the PBS washing step. Discard the supernatant and resuspend the cell pellets in 500 &#181;L of 4% paraformaldehyde fixation solution.</li>
	<li>After 10 min incubation at room temperature, centrifuge at 250 &#215; g, room temperature for 5 min. Discard the supernatant and wash cells with 4 mL of PBS.</li>
	<li>Repeat the PBS washing step. Discard the supernatant, resuspend the cell pellet in 200 &#181;L of saponin permeabilization buffer and transfer the suspension to a new 1.5 mL tube. After 10 min incubation at room temperature, centrifuge at 250 &#215; g, room temperature for 5 min.</li>
	<li>Discard the supernatant and resuspend the cell pellets in 200 &#181;L of primary antibody solution (1:500 &#947;H2AX antibody in antibody incubation solution). Incubate at 4 &#176;C overnight in the dark.</li>
	<li>Centrifuge the tubes at 250 &#215; g, 4 &#176;C for 5 min. Discard the supernatant and wash with 100 &#181;L of saponin wash solution.</li>
	<li>Repeat the washing step an additional two times. Discard the supernatant and resuspend the cell pellets in 500 &#181;L of EdU detection cocktail.</li>
	<li>Incubate at room temperature for 30 min in the dark. Centrifuge at 250 &#215; g, room temperature for 5 min. Discard the supernatant and wash with 1 mL of saponin wash solution.</li>
	<li>Repeat the washing step two times. Discard the supernatant and resuspend the cell pellets in 20-50 &#181;L of PBS. Proceed to section 4 for sample measurement.</li>
</ol>
4. Imaging senescence markers using the imaging flow cytometer system
<ol>
	<li>Empty the waste fluid bottle. Check the levels of speed beads, sterilizer, cleaner, debubbler, sheath, and rinse reagents (deionized water) to ensure sufficient fluids before turning on the instrument (see the Table of Materials).</li>
	<li>Turn on the instrument and imaging software (see the software interface in Figure 1A).</li>
	<li>Click on the Startup button to initialize fluidics and system calibration. 
	NOTE: This procedure takes approximately 45 min.</li>
	<li>Set the magnification to 40x, set fluidics speed to low, and turn on the lasers needed in the experiment. Turn on 405 nm, 488 nm, and 642 nm lasers to measure EdU-pacific blue, C12FDG, and Alexa Fluor 647-&#947;H2AX (or Alexa Fluor 647-Ki67), respectively. Set Ch6 for scatter channel, and Ch1 and Ch9 for brightfield.</li>
	<li>Start with the sample expected to have the highest fluorescence to set up the intensity of the lasers. Gently flip the sample tube to mix. Open the tube lid, and insert the sample tube onto the dock. Click on Load to start.</li>
	<li>Open a scatter plot and select the features Area_M01 and Aspect Ratio_M01 for the X- and Y-axes, respectively. Set a gate above aspect ratio of 0.5 to exclude doublets and cell aggregates below and on the right side, and the speed bead population on the left side (Figure 1B).</li>
	<li>Open a histogram plot and select the feature Gradient RMS_M01_Ch01 for the X-axis. Choose the singlet population and set the gate to select for focused cells (Figure 1C). 
	NOTE: The imaging system will automatically use speed beads to adjust the focus for imaging. However, it is recommended to select the right half of the histogram peak for the best-focused population.</li>
	<li>Open a histogram plot and select Raw Max Pixel Intensities for X-axis for each color channel (Ch2, 7, and 11). Adjust the laser powers (i.e., 488 nm, 405 nm, and 642 nm lasers for Ch2, 7, and 11, respectively), so each fluorochrome has a Raw Max Pixel value between 100 and 4,000 to avoid oversaturation. 
	NOTE: For this experiment, the laser power setting is 50 mW, 200 mW, and 50 mW for lasers 488 nm, 405 nm, and 642 nm, respectively.</li>
	<li>Choose the focused population to record and click on Acquire to measure the DLBCL samples with consistent settings. When changing samples for measurement, click on Return to recover the sample tube. Press Load to discard the sample.</li>
	<li>After all the samples are measured, turn off the brightfield and scatter laser. Measure single-color control samples to generate a compensation matrix.</li>
	<li>Click on Shutdown to close the imaging system.</li>
	<li>Analyze the data in the image analysis software.
	<ol>
		<li>Use the Spot Wizard tool of the image analysis software to automatically count and quantify nuclear &#947;H2AX foci in live-cell images. Select two cell populations (one with high and one with low spot count) to train the spot wizard for further automatic spot count analysis.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

This method examined the senescence-entering capability of four different DLBCL cell lines upon chemotherapy treatment, with bright-field imaging and flow cytometry-based quantification. On a single-cell level, we successfully detected major C12FDG+EdU-Ki67+ senescent populations in treated KARPAS422 and WSU-DLCL2 cells, and to a lesser extent in OCI-LY1 cells, while the SU-DHL6 cell line was resistant to the treatment. The difference in senescence-entering capability among cel...

A variety of stimuli can trigger cellular senescence, causing cells to enter a state of stable cell cycle arrest. These stimuli include intrinsic signaling changes or extrinsic stresses. Intrinsic signals include progressive telomere shortening, changes in telomere structure, epigenetic modification, proteostasis disorders, mitochondrial dysfunction, and activation of oncogenes. Extrinsic stresses include inflammatory and/or tissue damage signals, radiation or chemical treatment, and nutritional deprivation1,2,3,4. Among distinct types of senescence, the most commonly seen and well-studied are replicative senescence, oncogene-induced senescence (OIS), radiation-induced senescence, and therapy-induced senescence (TIS). OIS is an acute cellular response to genotoxic damage caused by replicative stress generated by aberrant oncogene activation and can to some extent prevent the pathological progression from a preneoplastic lesion to a full-blown tumor. TIS happens when tumor cells are stressed by chemotherapeutic drugs or ionizing radiation5,6.
Senescence is considered a double-edged sword in pathology due to its highly dynamic nature. It was initially described as a beneficial tumor-suppressive mechanism to remove damaged cells from the circulating pool of dividing cells, safeguarding the normal function of organs and inhibiting tumor growth7,8,9. However, emerging evidence has suggested a dark side of senescence. Senescent cells secrete proinflammatory cytokines, known as senescence-associated secretory phenotype (SASP), leading to fibrosis and malfunctional organs and promoting tumor initiation and progression10. Moreover, senescent cancer cells undergo epigenetic and gene-expression reprogramming in parallel with chromatin remodeling and activation of a sustained DNA-damage response (DDR)11,12, newly acquiring new cancer-stem-cell properties3. Although senescence-capable tumors respond better to therapeutic intervention compared to senescence-incapable ones13, the persistence of senescent cells may lead to a poor long-term prognosis if they are not effectively identified and eliminated by senolytic drugs5. Either way, a reliable method to assess senescence is of significant clinical interest, not only for the prognosis of therapy treatment but also for the development of novel strategies targeting senescent cells.
Regardless of different triggers, senescent cells exhibit some common features, including enlarged, flattened, multinucleated morphology with big vacuoles, significantly expanded nuclei, formation of H3K9me3-rich senescence-associated heterochromatin (SAHF) in the nucleus, persistent accumulation of DNA damage marker &#947;H2AX foci, activated p53-p21CIP1 and Rb-p16INK4a cell cycle regulatory mechanisms, stable G1 cell cycle arrest, massive induction of SASP, and elevated senescence-associated &#946;-galactosidase (SA-&#946;-gal) activity14. Since no single marker is sufficient to define senescence, enzymatic staining for SA-&#946;-gal activity, which is considered the gold standard for senescence detection, is usually combined with immunohistochemical staining for H3K9me3 and Ki67 to detect TIS15. However, chemical chromogenic-based SA-&#946;-gal is difficult to quantify. Here, we combined 5-dodecanoylaminofluorescein-di-&#946;-D-galactopyranoside (C12FDG) fluorescence-based SA-&#946;-gal (fSA-&#946;-gal) detection with immunofluorescent staining for &#947;H2AX and EdU-incorporated DNA to identify C12FDG+EdU-&#947;H2AX+ senescent cells using the advanced imaging flow cytometer system, which combines the speed, sensitivity and detailed single-cell images with spatial information that cannot be provided by flow cytometry and microscopy. This method enables rapid generation of high-resolution images allowing for the positioning and quantification of fluorescent signals within cells, while licensing the swift analysis of multiple samples by building standard pipelines.

<table><tbody><tr><td>Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody</td><td>Biolegend</td><td>613407</td><td></td></tr><tr><td>Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)</td><td>Biolegend</td><td>350509</td><td></td></tr><tr><td>C12FDG (5-Dodecanoylaminofluorescein Di-&amp;beta;-D-Galactopyranoside)</td><td>Fisher Scientific</td><td>11590276</td><td></td></tr><tr><td>Chloroquin -diphosphat</td><td>Sigma aldrich</td><td>C6628</td><td></td></tr><tr><td>Cleanser (Coulter Clenz)</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Click-iT EdU Pacific Blue Flow Cytometry Assay Kit</td><td>Thermo Scientific</td><td>C10418</td><td></td></tr><tr><td>Daunorubicin</td><td>Medchemexpress</td><td>HY-13062A</td><td></td></tr><tr><td>Debubbler (70% Isopropanol)</td><td>Millipore</td><td>1.3704</td><td></td></tr><tr><td>Image Analysis software (Amnis&amp;nbsp;IDEAS&amp;nbsp;6.3)</td><td>Luminex</td><td>CN-SW69-12</td><td></td></tr><tr><td>Instrument and imaging software (Amnis&amp;nbsp;ImageStreamX&amp;nbsp;Mk II Imaging Flow Cytometer System and INSPIRE software)</td><td>Luminex</td><td>100220</td><td></td></tr><tr><td>KARPAS</td><td>DSMZ</td><td>ACC 31</td><td></td></tr><tr><td>mafosfamide cyclohexylamine</td><td>Niomech</td><td>D-17272</td><td></td></tr><tr><td>OCI-LY1</td><td>DSMZ</td><td>ACC 722</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>11473704</td><td></td></tr><tr><td>PETG (2-Phenylethyl-&amp;beta;-D-thiogalactosid)&amp;nbsp;</td><td>Sigma aldrich</td><td>P4902</td><td></td></tr><tr><td>saponin</td><td>Sigma aldrich</td><td>47036</td><td></td></tr><tr><td>Sheath</td><td>Millipore</td><td>BSS-1006-B</td><td></td></tr><tr><td>SpeedBead Kit for ImageStream</td><td>Luminex</td><td>400041</td><td></td></tr><tr><td>Sterilizer (0.4-0.7% Hypochlorite)</td><td>VWR</td><td>JT9416-1</td><td></td></tr><tr><td>SU-DHL6</td><td>DSMZ</td><td>ACC 572</td><td></td></tr><tr><td>WSU-DLCL2</td><td>DSMZ</td><td>ACC 575</td><td></td></tr></tbody></table>

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.

A compensation matrix was generated using image analysis software by loading recorded data of single-color control samples. As shown in Supplemental Figure S1, a non-negligible (coefficient value &#8805; 0.1) light spillover from EdU to C12FDG was detected with crosstalk coefficient value 0.248, while the crosstalk among other channels was not significant. Four different DLBCL cell lines were treated with 5 &#181;g/mL MAF or 20 ng/mL DN to induce cellular senescence and analyzed using either c...

Watch this Scientific Journal Video about Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells at JoVE.com

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

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

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

simultaneous-imaging-flow-cytometry-based-detection-multiple

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

2.8K Views.  Johannes Kepler University Linz. Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

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

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Bioengineering

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Medicine

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunology and Infection

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Biology

Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

Source: Granier, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56606">Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an in-situ fluorescence multispectral imaging technique to analyze different subpopulations of infiltrating T cells in tumor tissue. A renal cell carcinoma tissue cryosection is treated with antibodies to label CD8+ T cells that express specific inhibitory receptors and exhibit an immunotherapy-resistant phenotype. The labeled section is visualized using a multispectral microscope to identify the distinct CD8+ T cell subpopulations.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. In Situ Immunofluorescence ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

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

Source: Flor, A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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 ...

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

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