Name: examination of pyroptosis by flow cytometry
Uploaded: 2024-05-31T14:40:00.000+00:00
Duration: 5 min 14 s
Description: Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...

We would like to thank Dr. Jue Liu for his kind assistance. This study was supported by grants from the National Key Research and Development Program of China (No. 2022YFD1800300), the National Natural Science Foundation of China (No. 32130105), and the Earmarked Fund for Modern Agro-Industry Technology Research System (No. CARS-40), China.

The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Preparation of sample cells
<ol>
	<li>Culture DF-1 (immortal chicken embryo fibroblasts) cells in six-well plates (5 x 105 cells per well) with Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 38 &#176;C.</li>
	<li>When the cells reach 80% confluence, discard the serum-containing cell culture medium, wash the cells three times with phosphate-buffered saline (PBS), and then add 2 mL of serum-free cell culture medium to each well.</li>
	<li>Add the IBDV virus solution (containing calculated MOI) to each well, and set up a mock-infected control by adding the volume of DMEM equal to that of the virus solution.</li>
	<li>After 1 h of virus adsorption, discard the culture medium, wash the cells with PBS three times, add 2 mL of DMEM supplemented with 2% FBS to each well, and continue cell cultures for 24 h.</li>
	<li>24 h post-IBDV infection, perform trypsin treatment (500 &#181;L per well) for 1 min and subsequently neutralize by DMEM supplemented with 10% FBS (2 mL per well). Afterward, the cells must be harvested to prepare single-cell suspensions for flow cytometry.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at 2-8 &#176;C. Discard the supernatant using a pipette.</li>
	<li>Resuspend the cell pellet in PBS and gently vortex the tubes for 3 s.</li>
	<li>Centrifuge the cells as described in step 1.6 (400 x g for 5 min at 2-8 &#176;C).</li>
	<li>Repeat the wash of cells as described in step 1.7 and step 1.8.</li>
	<li>Perform cell counting using a hemocytometer under a microscope. Then, resuspend the cells in an appropriate volume of flow cytometry staining buffer (so that the final cell concentration is 1 x 107 cells/mL). Gently vortex the suspensions.</li>
</ol>
2. Cell staining for flow cytometry
<ol>
	<li>Add 100 &#181;L of the single-cell suspensions from step 1.10 into 5 mL round-bottom polystyrene tubes (12 mm &#215; 75 mm). Prepare mock- and IBDV-infected cells for anti-chGSDME-NT/CT antibody staining, and use normal mouse IgG as an isotype control. Meanwhile, prepare blank control and single stained tubes with IBDV-infected cells.</li>
	<li>Add 1 &#181;g of either Alexa Fluor 647-labeled anti-chGSDME-NT McAb, anti-chGSDME-CT McAb, or normal mouse IgG (as an isotype control) to IBDV-infected and mock-control tubes, respectively. Subsequently, gently vortex the tubes for 3 s.</li>
	<li>Incubate the stained cells at 2-8 &#176;C or on ice for 30 min in the dark. Gently vortex tubes every 10 min.</li>
	<li>Wash the cells with 1 mL of flow cytometry staining buffer by centrifugation at 400 x g for 5 min at 2-8 &#176;C. Discard the supernatant using a pipette.</li>
	<li>Repeat the wash of cells as described in step 2.4.</li>
	<li>Resuspend the pellet in 100 &#181;L of flow cytometry staining buffer.</li>
	<li>Stain the cells of the IBDV-infected group and mock-infected group, as well as the PI-single stained tubes, with 10 &#181;g/mL of PI for 10 min at room temperature, followed by adding 400 &#181;L of flow cytometry staining buffer for the flow cytometry assay.</li>
</ol>
3. Conducting the flow cytometry assay
<ol>
	<li>Turn on the flow cytometer to initialize the instrument and launch the software.</li>
	<li>Begin with running the blank control tube followed by the single stained tubes to adjust the voltage and compensation parameters of the flow cytometer. Utilize FL3 and FL4 fluorescence channels, which emit at 635 nm, for detecting PI and Alexa Fluor 647 fluorophores, respectively.</li>
	<li>After configuring all the parameters, run all the samples sequentially.</li>
</ol>
4. Analysis of flow cytometry data
<ol>
	<li>Analyze the Alexa Fluor 647 staining of each sample in the FL4 channel histogram.</li>
	<li>To assess the proportion of double-positive cells in each sample, utilize the four-quadrant dot plot of FL3/FL4.</li>
	<li>Set positive thresholds based on isotype control IgG/PI dual-stained samples, ensuring that the proportion of double-positive cells remains below 1%. This approach facilitates measuring the proportion of double-positive cells in all samples.</li>
</ol>

Quantifying Pyroptosis in Infectious Bursal Disease Virus-Infected DF-1 Cells

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C., Ni&#241;o-Fong, R., Lopez, A., Frederick Markham, R. J., Kibenge, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+bursal+disease+virus+(IBDV)+induces+apoptosis+in+chicken+B+cells.">Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells.</a> Comp Immunol Microbiol Infect Dis. 28 (4), 321-337 (2005).</li><li>Wang, S., Liu, Y., Zhang, L., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+monitoring+cancer+cell+pyroptosis.">Methods for monitoring cancer cell pyroptosis.</a> Cancer Biol Med. 19 (4), 398-414 (2021).</li><li>Zhang, Y., Chen, X., Gueydan, C., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+changes+during+programmed+cell+deaths.">Plasma membrane changes during programmed cell deaths.</a> Cell Research. 28 (1), 9-21 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyroptosis+is+driven+by+non-selective+gasdermin-d+pore+and+its+morphology+is+different+from+mlkl+channel-mediated+necroptosis.">Pyroptosis is driven by non-selective gasdermin-d pore and its morphology is different from mlkl channel-mediated necroptosis.</a> Cell Research. 26 (9), 1007-1020 (2016).</li></ol>

The authors have nothing to disclose.

This article describes an effective method for examining pyroptosis using flow cytometry, achieved through dual staining of infected cells with Alexa Fluor 647-labeled anti-chGSDME-NT McAb and PI. This approach can also be applied across various cell types to differentiate pyroptosis from other types of cell death, such as apoptosis and necrosis.
Pyroptosis, an inflammatory type of programmed cell death primarily reliant on Gasdermin (GSDM) D-induced plasma membrane pore formation in mammals1,2,3, has traditionally been assessed through methods like LDH release6,15, morphological features16, and detection of GSDM-NT/GSDM-CT fragments via Western Blot or IFA assays17. However, none of these methods accurately quantifies the percentage of pyroptotic cells. This study leverages the characteristic plasma membrane pores in pyroptotic cells formed by GSDM-NT fragments to develop a flow cytometry-based strategy for their determination.
When cells were dual-stained with chGSDME-NT-Alexa Fluor 647 and PI and subsequently analyzed via flow cytometry in the FL3 and FL4 fluorescence channels, pyroptosis was discernible via two parameters concurrently: chGSDME-NT-Alexa Fluor 647 staining indicating plasma membrane pore formation by the N-terminal fragment of cleaved chGSDME, and PI staining indicating plasma membrane rupture. The data demonstrate that the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (approximately 33%) was significantly higher than in mock-infected controls (P &#60; 0.001).
The experimental method requires several considerations to ensure accuracy and reliability. Firstly, each sample tube used for flow analysis should contain no fewer than 1 x 106 cells to maintain the precision of the flow analysis. Given the multiple washing steps in the protocol, it is advisable to increase the initial number of cells in each tube at step 2.1 accordingly, for example, to 2 x 106 cells. It&#39;s crucial to handle cell washing and transferring with extreme caution to avoid unintended disruption of cell integrity. Additionally, keep in mind that cell density, antibody concentration, and the duration of the operation can all influence the optimal parameter settings. Therefore, it&#39;s necessary to adjust the parameters and settings of the flow cytometer based on the specific experimental conditions. In this study, the parameters used were as follows: FSC AmpGain was set to 1.40, and SSC voltage was set to 490 V. The gate in the FSC/SSC plot was set to cover all cell populations for subsequent FL3/4-channel analysis. Fluorescence voltages were set to 600 V for the FL3 channel (PI) and 600 V for the FL4 channel (Alexa Fluor 647). Compensation was set to 38.0% between the FL4 and FL3 channels. The flow rate was set to 200 events/s, with an event threshold set at 10,000.
Assisted by the novel approach, this comprehension of the role of GSDME-mediated pyroptosis in the host response to pathogenic infections in chickens and the underlying mechanisms has significantly advanced. Through the use of specific antibodies, we were able to examine pore-forming chGSDME-NTvia flow cytometry, distinguishing pyroptotic cells from others and facilitating the determination of the proportion of cells undergoing pyroptosis. As a result, this method offers a distinct advantage over Western Blot or IFA assays in detecting pyroptotic cells.
However, the limitation of this protocol lies in using the N-terminal fragment of chicken GSDME in IBDV-infected cells as an experimental model for studying pyroptosis. Nonetheless, this study marks the first successful utilization of flow cytometry for determining pyroptosis.
In summary, the flow cytometry method described here for examining pyroptosis holds great potential for advancing our understanding of the mechanisms behind pyroptosis in both mammals and avians. This method stands as an effective tool that contributes to research on cell death in life sciences.

Pyroptosis is an inflammatory type of programmed cell death that mainly depends on the formation of plasma membrane pores by Gasdermin (GSDM) D in mammals1,2,3. Due to the genetic deficiency of GSDMD in chickens4,5, the mechanism of pyroptosis in chickens remains elusive. The Gasdermin family comprises conserved proteins, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB593,6. Studies have reported that GSDME from teleost fish and ducks is cleaved by caspase-1/3/7 or caspase-3/7 to induce pyroptotic cell death7,8. However, the role of GSDME-mediated pyroptosis in the host response to pathogenic infections in chickens remains to be elucidated.
Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive poultry disease caused by IBDV9. IBDV, a non-enveloped bi-segmented double-stranded (ds) RNA virus, belongs to the genus Avibirnavirus in the Birnaviridae family10. Previous studies by others and our laboratory have shown that IBDV infection induces cell death in host cells via different pathways11,12,13,14. Previous findings have demonstrated that IBDV infection triggers the release of lactate dehydrogenase (LDH), an indicator of lytic cell death6,15, suggesting that IBDV infection induces lytic cell death in host cells. Furthermore, the data show that IBDV-infected cells exhibit morphological features of pyroptotic cell death, including cell swelling with large bubbles blowing from the plasma membrane and propidium iodide (PI)-staining positive, suggesting that IBDV infection induces pyroptosis in cells.
Considering that the formation of membrane pores in pyroptotic cells by the N-terminal fragment of cleaved GSDM (GSDM-NT) is a hallmark of pyroptosis, theoretically, pyroptotic cells could be detected by flow cytometry by examining GSDM-NT on the cell membrane using specific antibodies. In avian pyroptotic cells, the N-terminal fragment of chicken Gasdermin E (chGSDME-NT) forms membrane pores, allowing propidium iodide (PI) to pass through and bind DNA. Thus, the proportion of pyroptotic cells can be detected using flow cytometry, thereby distinguishing pyroptosis from other forms of cell death, such as apoptosis and necrosis. However, the method for examining pyroptotic cells by flow cytometry has not been reported. In this study, DF-1 cells were infected with IBDV, and flow cytometry was conducted to examine pyroptotic cells using monoclonal antibodies (McAb) against the chGSDME-NT fragment (membrane-bound) and PI staining. Surprisingly, pyroptotic cells were effectively detected by flow cytometry. Furthermore, the proportion of pyroptotic cells could be quantified. These findings provide a powerful and effective means to determine pyroptosis.
This article describes a method for examining pyroptosis in IBDV-infected cells by flow cytometry using anti-chGSDME-NT McAb and PI staining. This method can also be extended to other pathogen-infected cells and applied to examine various cell types with pyroptosis, distinguishing them from other forms of cell death.

<table><tbody><tr><td>5 mL round-bottom polystyrene tube (12 &amp;times; 75 mm)</td><td>Corning Falcon</td><td>352052</td><td></td></tr><tr><td>6 Well Cell Culture Plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Alexa Fluor 647 antibody labeling kits</td><td>Thermo Fisher Scientific</td><td>A20186</td><td></td></tr><tr><td>Anti-chGSDME-CT McAb</td><td>SAE Biomedical Tech Company (Zhongshan, China)</td><td>EU0228</td><td></td></tr><tr><td>Anti-chGSDME-NT McAb</td><td>SAE Biomedical Tech Company (Zhongshan, China)</td><td>EU0227</td><td></td></tr><tr><td>CellQuest software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific</td><td>3100</td><td></td></tr><tr><td>Cryogenic Freezing Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>C11995500BT</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F0193-500ML</td><td></td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>FACSCalibur</td><td></td></tr><tr><td>Flow Cytometry Staining Buffer</td><td>Thermo Fisher Scientific</td><td>00-4222-26</td><td></td></tr><tr><td>Hemocytometer</td><td>Qiu-jing Biochemical Reagent &amp;amp; Instrument Company (Shanghai, China)</td><td>YX-JSB52</td><td></td></tr><tr><td>IBDV Lx strain</td><td></td><td></td><td>IBDV Lx strain was kindly provided by Dr. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China</td></tr><tr><td>Inverted Microscope</td><td>Chongqing Photoelectric Instrument Company</td><td>XDS-1B</td><td></td></tr><tr><td>Normal Mouse IgG</td><td>Santa Cruz Biotechnology</td><td>sc-2025</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>M&amp;amp;C&amp;nbsp; Gene Technology</td><td>CC017</td><td></td></tr><tr><td>Propidium Iodide(PI)</td><td>Sigma-Aldrich</td><td>P4170</td><td></td></tr><tr><td>Trypsin-EDTA, 0.25%</td><td>M&amp;amp;C&amp;nbsp; Gene Technology</td><td>CC008</td><td></td></tr><tr><td>Vortex Oscillator</td><td>MIULAB</td><td>MIX-28+</td><td></td></tr></tbody></table>

examination of pyroptosis by flow cytometry

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P &#60; 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

chGSDME-NT on the membrane of DF-1 cells with IBDV infection could be readily detected by flow cytometry 
One of the most important features of pyroptotic cells is the formation of membrane pores by GSDM-NT fragments generated from Gasdermin cleavage. Therefore, pyroptotic cells could theoretically be detected by flow cytometry via examining GSDM-NT on cell membranes using specific antibodies. Thus, DF-1 cells were infected with IBDV, and the pyroptotic cells were examined by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT McAb and PI staining. A considerable number of chGSDME-NT positive cells were detected by flow cytometry after IBDV infection (Figure 1). In contrast, mock-infected controls or cells stained with isotype control IgG showed minimal Alexa Fluor 647-positive cells. These findings demonstrate the effective and specific detection of membrane-bound chGSDME-NT by flow cytometry. Of note, as the C-terminal fragments of cleaved chGSDME (chGSDME-CT) were retained inside the cytoplasm rather than translocated to the plasma membrane like chGSDME-NT, chGSDME-CT fragments were barely detectable by flow cytometry using membrane surface antigen staining, which was in agreement with the fact that pyroptosis was associated with membrane-bound chGSDME-NT fragments.
Pyroptosis in DF-1 cells can be determined by flow cytometry using dual staining of cells with fluorescein-labeled anti-chGSDME-NT McAb and PI 
When cells were dual stained with Alexa Fluor 647-labeled anti-chGSDME-NT McAb and PI, followed by flow cytometry analysis in the FL3 and FL4 fluorescence channels, pyroptotic cells could be determined by chGSDME-NT and PI staining being positive. As shown in Figure 2A,B, a considerable number of chGSDME-NT positive cells with IBDV infection were detected by flow cytometry assays, and the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than that of mock-infected controls (P &#60; 0.001), suggesting that the formation of membrane pores by the chGSDME-NT was synchronous with the disruption of cell membranes, thereby causing these IBDV-infected cells to undergo pyroptotic cell death. These data clearly show that IBDV infection induces chGSDME-mediated pyroptosis in host cells. Whereas PI single-positive cells may represent other forms of cell death induced by IBDV infection. Therefore, examination of pore-forming chGSDME-NT by flow cytometry provides a valuable method for distinguishing pyroptotic cells from those undergoing other forms of cell death.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66912/66912fig01.jpg" /> 
Figure 1: Examination of chGSDME-NT bound cells by flow cytometry. DF-1 cells were mock infected or infected with IBDV at an MOI of 1. 24 h after infection, cells were harvested and stained with Alexa Fluor 647-labeled anti-chGSDME-NT /anti-chGSDME-CT McAb or isotype control IgG, and analyzed with &#64258;ow cytometry. chGSDME-NT-staining positive in flow cytometry assay demonstrated the membrane-bound chGSDME-NT cells with IBDV-induced pyroptosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66912/66912fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66912/66912fig02.jpg" /> 
Figure 2: Examination of pyroptosis by flow cytometry using dual staining with chGSDME-NT and PI. (A) Dot-plot analysis of pyroptotic cells by flow cytometry. DF-1 cells were infected with IBDV at an MOI of 1. 24 h after infection, cells were harvested, stained with Alexa Fluor 647-labeled anti-chGSDME-NT /anti-chGSDME-CT McAb and PI, and analyzed with flow cytometry. (B) The percentage of pyroptotic cells in each group in (A) was plotted and statistically analyzed. Two-way ANOVA was employed to analyze statistical significance. The data represented three independent experiments and were presented as means &#177; the SD. ***P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66912/66912fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Flow Cytometric Determination of Pyroptosis in Avian Cells

This article describes the identification of pyroptotic cells using flow cytometry after dual staining with antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI).

Examination of Pyroptosis by Flow Cytometry

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated ...

examination-of-pyroptosis-by-flow-cytometry

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

924 Views.  National Key Laboratory of Veterinary Public Health Security. Het doel van ons onderzoeksteam is om te bepalen of vogelcellen pyroptose ondergaan die vergelijkbaar is met die van zoogdiercellen. Bovendien streven we ernaar om pyroptose op nauwkeurigere en eenvoudigere wijze vast te stellen. Veel genen in vogelsoorten verschillen aanzienlijk van die in zoogdieren, waardoor het moeilijk is om reagentia te gebruiken die vaak worden gebruikt in zoogdiercelstudies voor onderzoek naar vogelceldood.Het onderzoek naar vo...

Video: Auteur in de schijnwerpers: flowcytometrische bepaling van pyroptose in vogelcellen

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Cancer Research

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

FACS Analysis of Lipid Hydroperoxides in Ferroptotic Medulloblastoma Cells

Revealing the Ferroptotic Phenotype of Medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named &#34;ferroptosis.&#34; The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic &#34;bubbling&#34; phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics 

Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Neuroscience

Quality-Controlled Sputum Analysis by Flow Cytometry

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs.
The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

This protocol describes an efficient method for dissociating sputum into a single cell suspension and the subsequent characterization of cellular subsets on standard flow cytometric platforms.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Examination of Pyroptosis by Flow Cytometry

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