Name: neutrophil lifespan extension with clon-g and an in vitro spontaneous death assay
Uploaded: 2023-05-12T13:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay at JoVE.com

This project was supported by the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00036, 22HHXBSS00019), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2021-I2M-1-040&#65292;2022-I2M-JB-015), the Special Research Fund for Central Universities, Peking Union Medical College (3332022062), and the Science and Technology Support Program of Sichuan Province (NO. 2021YJ0480).

The Boston Children&#39;s Hospital and State Key Laboratory of Experimental Hematology (SKLEH) Animal Care and Use Committee approved and monitored all the procedures. Figure 1 depicts a flow chart of neutrophil culturing with CLON-G and the in vitro death assay.
1. Neutrophil lifespan extension with CLON-G
NOTE: All the mentioned operations and materials must be sterile. Ensure all the solutions are well mixed and distributed evenly.
<ol>
	<li>Preservation of the CLON-G components
	<ol>
		<li>Dissolve 50 mg of Q-VD-oph (see Table of Materials) to a final concentration of 100 mM by adding 973.7 &#181;L of dimethyl sulfoxide (DMSO), and pipette the liquid until the mixture becomes clear. Aliquot 50 &#181;L per tube, and preserve at &#8722;20 &#176;C. 
		CAUTION: DMSO is a harmful chemical liquid. Wear a lab coat, goggles, and a mask to avoid skin contact, eye contact, and inhalation.</li>
		<li>Thaw Hsp70 (see Table of Materials) at 4 &#176;C, and spin down the contents in the vial. Aliquot 1 &#181;L of Hsp70 per tube on ice, and preserve at &#8722;80 &#176;C. 
		NOTE: Hsp70 is a protein; ultra-cold storage is necessary to keep it stable. Avoid the freeze-thaw cycle.</li>
		<li>Dissolve 1 mg of DFO (see Table of Materials) with 7.61 mL RPMI 1640 to obtain a stock of 200 &#181;M. Aliquot 500 &#181;L per tube, and preserve at &#8722;20 &#176;C.</li>
		<li>Dissolve 0.1 g of NAC (see Table of Materials) with 2.5 mL of RPMI 1640 in a 15 mL centrifuge tube, and adjust the pH to 7-7.4 with NaOH. Add RPMI 1640 to a final volume of 3.06 mL to make a 200 mM NAC stock. Filter it with a 0.2 &#181;m filtering unit. Aliquot 500 &#181;L per tube, and preserve at &#8722;20 &#176;C. 
		NOTE: Dissolving NAC in this high concentration is not easy, and vortexing can help dissolve it. Phenol red in RPMI 1640 is a perfect indication of the pH; when the NAC is just dissolved, the color is yellowish; adjust it until it becomes pinkish. NAC stock solutions are stable for up to 1 month at &#8722;20 &#176;C.</li>
		<li>Dissolve 10 mg of Nec-1s (see Table of Materials) to 20 mM with 1.8 mL of DMSO, and pipette the mixture until it becomes clear. Aliquot 50 &#181;L per tube, and preserve at &#8722;20 &#176;C. Protect from light.</li>
		<li>Dissolve 250 &#181;g of G-CSF (see Table of Materials) to 200 &#181;g/mL with 1.25 mL of RPMI 1640. Aliquot 50 &#181;L per tube, and preserve at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of 2x CLON-G culture medium
	<ol>
		<li>Prepare the basic medium. Add 39.5 mL of RPMI 1640, 10 mL of fetal bovine serum (FBS), and 0.5 mL pen-strep (antibiotics) to a 50 mL tube to make RPMI 1640 with 20% FBS and 1% PS as a basic medium. 
		NOTE: This high concentration of FBS is used to provide for the prolonged culture of neutrophils, which might be greater than 5 days. If the culturing time is 1-2 days, 10%-15% FBS is sufficient.</li>
		<li>Thaw all the stock solutions of the CLON-G components on ice.</li>
		<li>Dilute 1 &#181;L of Hsp70 to 20 &#181;M by adding 606 &#181;L of the basic medium. Dilute 1 &#181;L of 200 &#181;g/mL G-CSF to 20 &#181;g/mL by adding 9 &#181;L of the basic medium.</li>
		<li>Add 976 &#181;L of basic medium to a 15 mL centrifuge tube. Add 1 &#181;L of Q-VD-oph (100 mM), 10 &#181;L of DFO (200 &#181;M), 1 &#181;L of Hsp70 (20 &#181;M), 10 &#181;L of NAC (200 mM), 1 &#181;L of Nec-1s (20 mM), and 1 &#181;L of G-CSF (20 &#181;g / mL) to make 1 mL of 2x CLON-G medium. 
		NOTE: The 2x CLON-G medium should be used immediately after it is prepared. One may store the 2x CLON-G temporarily at 4 &#176;C. Leftover 20 &#181;M Hsp70 must be discarded.</li>
	</ol>
	</li>
	<li>Neutrophil culture
	<ol>
		<li>Isolate mouse neutrophils by gradient centrifugation following a previous report25. Resuspend the isolated mouse neutrophils in the basic medium (1 million cells/100 &#181;L). 
		NOTE: Avoid activating the neutrophils by handling them gently. Avoid foaming during the whole process.</li>
		<li>Add 500 &#181;L of 2x CLON-G medium, 400 &#181;L of the basic medium, and 100 &#181;L of the cell suspension to 24-well non-tissue cultured culture plates (see Table of Materials), and gently pipette three to four times. 
		NOTE: High concentrations of the components in the 2x CLON-G medium might damage the neutrophils; avoid adding them together without the basic medium. The non-tissue cultured culture plate is necessary to avoid neutrophil adhesion.</li>
		<li>Place the culture plate in a 37 &#176;C and 5 % CO2 incubator smoothly. 
		NOTE: It is unnecessary to exchange the cell medium within 7 days. The final concentrations of the CLON-G composition are 50 &#181;M Q-VD-Oph, 1 &#181;M DFO, 10 pM Hsp70, 1 mM NAC, 10 &#181;M Nec-1 s, and 10 ng/mL G-CSF.</li>
		<li>Stain the cultured neutrophils with Wright Giemsa compound stain (see Table of Materials), and analyze with a light microscope (Figure 2A-D). Alternatively, stain with APC-CD11b and PE-Cy7-Ly6G antibodies, and analyze with flow cytometry (Figure 2E-I) as described previously26.</li>
	</ol>
	</li>
</ol>
2. In vitro spontaneous death assay of neutrophils
<ol>
	<li>Flow cytometry analysis
	<ol>
		<li>Culture isolated neutrophils at a density of 1 million cells/mL in the basic medium at 37 &#176;C and 5% CO2. The 24-well culture plate is non-tissue cultured. Add 1 mL of cell medium per well.</li>
		<li>Dissolve 1 g of CaCl2 with 45 mL of saline to make 200 mM CaCl2. Filter it with a 0.2 &#181;m filtering unit. Preserve at 4 &#176;C.</li>
		<li>Prepare the staining mix. Add 7 &#181;L of saline per sample to a 1.5 mL tube, and add 0.4 mg/mL FITC-Annexin-V, 0.5 mg/mL propidium iodide (PI), and 200 mM CaCl2 solution at 1 &#181;L each per sample. Mix well. Use the solution immediately after preparation.</li>
		<li>Pipette the cell medium gently five to seven times to mix well, followed by transferring 100 &#181;L to a flow cytometry tube at the desired time points. 
		NOTE: Avoid foaming during the whole process.</li>
		<li>Vortex counting beads (see Table of Materials) to mix them well. Pipette 10 &#181;L of the well-mixed counting beads to the same flow cytometry tube as in step 2.1.4.</li>
		<li>Add 10 &#181;L the prepared staining mix (step 2.1.3) to the flow cytometry tube. Incubate at room temperature for 5 min away from light.</li>
		<li>Add 100 &#181;L of saline to the tube, and mix well to ensure the even distribution of the counting beads and cells.</li>
		<li>Perform flow cytometry analysis of the cell medium. Collect the cells at a low rate with ~400 events/s. Gate the APC-PE-Cy7- cells to FSC-A and SSC-A, and gate the intact cells with medium FSC-A and SSC-A positions to the FITC-Annexin-V and PE-PI; the Annexin-V&#8722;PI&#8722; population is classified as healthy cells. 
		NOTE: The following equations determine the total number of healthy cells per sample and the viability of the neutrophils: 
		Total number of healthy cells per sample27 = (1,000/number of counting beads) &#215; number of Annexin-V&#8722;PI&#8722; population &#215; 10 
		&#8203;Viability of neutrophils = total number of healthy cells at the indicated time/total number of healthy cells at time zero</li>
	</ol>
	</li>
	<li>Fluorescent image assay
	<ol>
		<li>Culture isolated neutrophils at a density of 1 million cells/mL in the basic medium at 37 &#176;C and 5% CO2 in a confocal plate with 2 mL of cell medium per plate. 
		NOTE: A confocal fluorescence microscope has the best image quality, but any fluorescence microscope that has 488/561/DIC channels is sufficient to support this experiment.</li>
		<li>Take the plate out gently at the indicated time point. Add 10 &#181;L each of 0.4 mg/mL FITC-Annexin-V, 0.5 mg/mL PI, and 200 mM CaCl2 to the plate evenly. Stain at room temperature for 5 min. 
		NOTE: Avoid shaking during the whole process. Add the staining agents evenly and gently.</li>
		<li>Capture fluorescence images at 488 (annexin-V)/561 (PI)/DIC channels using a confocal fluorescence microscope with a 20x objective lens (see Table of Materials). Set the exposure time of the 488 channel as 500 ms, the 561 channel as 200 ms, and the DIC channel as 100 ms. Select 5-10 random areas for each plate. The cell types are defined in our previously published report14.</li>
	</ol>
	</li>
</ol>

The authors declare that the research was conducted in the absence of any conflicts of interest.

Neutrophils play vital roles in innate and adaptive immunity, and their homeostasis is tightly regulated. Neutrophils are the most abundant leukocytes in human peripheral blood, and they have a robust and fast turnover. A healthy adult can release 1 x 109 neutrophils/kg daily from the bone marrow28. The death of neutrophils has hence become one of the puzzling enigmas of this field, and much effort has been dedicated to better understanding them. Caspase29,<...

Neutrophils are known to comprise an arsenal of abundant cytoplasmic granules, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, antimicrobial enzymes, and various organelles that defend against invading microbes; additionally, they are highly motile and are the first cells recruited to the inflammation site, meaning neutrophils are the first line of defense of the innate immune system1,2. Granulocyte transfusion therapy has hence become a promising clinical treatment for neutropenia-related infections to transiently boost neutrophil immunity3,4,5. Recent discoveries have clearly shown that neutrophils also function as multifaced effectors in many physiopathological scenarios6. The average lifespan of a neutrophil is less than 24 h, and, thus, basic research on neutrophils and the application of neutrophil studies are tremendously difficult due to the limitations related to stable genetic manipulation and long-term storage7,8,9,10,11. There are some cell lines that can partially showcase some neutrophil functions, such as HL-60, PLB-985, NB4, Kasumi-1, and induced pluripotent stem cells12. These cell lines can achieve effective gene editing and cryopreservation; however, they still differ quite immensely from primary neutrophils and, thus, cannot faithfully recapitulate neutrophil functions13. Thus, most of the research in this field still relies on freshly isolated primary neutrophils. The field still relies on generating expensive and time-consuming conditional knock-out mice to investigate specific gene functions in neutrophils, but no human models currently exist.
Having put our effort into exploring the heterogeneous processes involved in neutrophil death and the multiple pathways that regulate these processes14,15, a new treatment termed CLON-G (caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor) was recently reported16. CLON-G consists of Q-VD-oph (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), Hsp70 (heat shock protein 70), DFO (deferoxamine), NAC (N-acetylcysteine), Nec-1s (necrostatin-1s), and G-CSF (granulocyte colony-stimulating factor). Neutrophil spontaneous death is mediated by multiple pathways, including apoptosis, necroptosis, and pyroptosis. Q-VD-oph inhibits the apoptosis of neutrophils as a pan-caspase inhibitor by targeting caspase 1, caspase 3, caspase 8, and caspase 917. Neutrophil necroptosis is dependent on a signaling pathway involving receptor-interacting protein kinase-1 (RIPK1) and mixed lineage kinase domain-like protein (MLKL)18. As an RIPK1 inhibitor, Nec-1s inhibit the necroptosis of neutrophils. Hsp70 and DFO can inhibit lysosomal membrane permeabilization (LMP), which could induce neutrophil apoptosis19 and pyroptosis20. Reactive oxygen species (ROS) play vital roles in neutrophil death by mediating LMP19 and apoptosis21 and by inhibiting survival signals22. As an antioxidant that can reduce ROS accumulation, NAC delays neutrophil death. As a growth factor, G-CSF activates neutrophil survival signals and inhibits calpain-induced apoptosis23,24. By simultaneously targeting multiple neutrophil spontaneous death pathways, the neutrophil lifespan can be effectively extended to greater than 5 days without compromising their function. CLON-G treatment expands the possibilities of neutrophil preservation, transportation, and gene manipulation, which can accelerate research in the neutrophil community. Meanwhile, based on the knowledge of neutrophil death, the currently approved protocols for cell death assays can cause unexpected damage to neutrophils14, so these protocols have been refined to be more appropriate for neutrophil studies. This report provides detailed protocols for neutrophil culturing with CLON-G and an in vitro cell death assay of mouse neutrophils using flow cytometry and fluorescence imaging. CLON-G is effective on both mouse and human neutrophils; however, the mouse samples are demonstrated here to simplify this protocol. The concentration of NAC is 1 mM for mouse neutrophils and 10 &#181;M for human neutrophils. Hsp70 is species-specific and, thus, should be utilized according to the source of the neutrophil. For this protocol, it does not matter whether neutrophils are isolated from peripheral blood or bone marrow and how they are isolated.
For the present study, neutrophils were isolated from mouse bone marrow to achieve enough neutrophils for the experiments, as about 1 x 107-1.5 x 107 neutrophils can be obtained from the bone marrow, while only 1 x 106 neutrophils can be isolated from the peripheral blood of a single 8-12 week old C57BL/6 mouse (of either sex). Gradient centrifugation was conducted to avoid possible damage and activation from the mechanical stimulation of FACS sorting or MACS sorting.

<table><tbody><tr><td>0.2 &amp;micro;m syringe filter</td><td>Pall Corporation</td><td>4612</td><td>Filtrate prepared CLON-G components.</td></tr><tr><td>1.5 mL micro centrifuge tube</td><td>LABSELECT</td><td>MCT-001-150</td><td>Lab consumable.</td></tr><tr><td>15 mL Centrifuge Tubes</td><td>LABSELECT</td><td>CT-002-15</td><td>Lab consumable.</td></tr><tr><td>24 well cell culture plate</td><td>Falcon</td><td>351147</td><td>Neutrophil culture plate.</td></tr><tr><td>50 mL Centrifuge Tubes</td><td>LABSELECT</td><td>CT-002-50</td><td>Lab consumable.</td></tr><tr><td>BD LSRII</td><td>BD</td><td></td><td>Instrument for flow cytometry analysis of neutrophil death.</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>C4901</td><td>Assitant of Annexin-V binding&amp;nbsp; to phosphatidylserine.</td></tr><tr><td>Confocal microscope</td><td>Perkinelmer</td><td>UltraVIEW VOX</td><td>Instrument for fluorescent analysis of neutrophil death.</td></tr><tr><td>Confocal plate</td><td>NEST</td><td>801001-20mm</td><td>Lab consumable for fluorescent image assay.</td></tr><tr><td>Counting beads</td><td>Thermo Fisher</td><td>C36950</td><td>Quantification in flow cytometry analysis of neutrophil death.</td></tr><tr><td>DFO</td><td>Sigma Aldrich</td><td>D9533</td><td>Component of CLON-G. LMP inhibitor.</td></tr><tr><td>Dimethyl sulfoxide ( DMSO)</td><td>Sigma Aldrich</td><td>D2650</td><td>Solvent for Q-VD-oph and Nec-1s.</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10099141C</td><td>Component of neutrophil culture basic medium. Nutrition supply.</td></tr><tr><td>FITC-Annexin-V</td><td>BD</td><td>51-65874X</td><td>Annexin-V can bind to phosphatidylserine of aged cells.This is at FITC channel.</td></tr><tr><td>Hsp70</td><td>Abcam</td><td>ab113187</td><td>Component of CLON-G. LMP inhibitor.</td></tr><tr><td>NAC</td><td>Sigma Aldrich</td><td>A9165</td><td>Component of CLON-G. Antioxidant.</td></tr><tr><td>Nec-1s</td><td>EMD Millipore</td><td>852391-15-2</td><td>Component of CLON-G. Necroptosis inhibitor.</td></tr><tr><td>Penicillin-Streptomycin Solution (PS)</td><td>Gibco</td><td>15070063</td><td>Component of neutrophil culture basic medium. Antibiotics to protect cells from bacteria comtamination.</td></tr><tr><td>Propidium Iodide (PI)</td><td>BioLegend</td><td>421301</td><td>For neutrophil death assay. A small fluorescent molecule that binds to DNA&amp;nbsp; but cannot passively traverse into cells that possess an intact plasma membrane.</td></tr><tr><td>Q-VD-oph</td><td>Selleck chem</td><td>S7311</td><td>Component of CLON-G. Pan-caspase inhibitor.</td></tr><tr><td>Recombinant Human Granulocyte Colony-stimulating Factor for Injection (CHO cell)(G-CSF)</td><td>Chugai Pharma China</td><td>GRANOCYTE</td><td>Component of CLON-G.&amp;nbsp; Promote neutrophil survival through Akt pathway.</td></tr><tr><td>Round-Bottom Polystyrene Tubes</td><td>Falcon</td><td>100-0102</td><td>Lab consumable for flow cytometry analysis.</td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>C11875500BT</td><td>Component of neutrophil culture basic medium.</td></tr><tr><td>Saline</td><td>LEAGENE</td><td>R00641</td><td>Solution for flow cytometry analysis of neutrophil death.</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>FENG CHUAN</td><td>13-011-00029</td><td>pH adjustion for NAC.</td></tr><tr><td>Wright-Giemsa Stain Solution</td><td>Solarbio</td><td>G1020</td><td>Neutrophil cytospin staining.</td></tr></tbody></table>

neutrophil lifespan extension with clon-g and an in vitro spontaneous death assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

The Wright-Giemsa-stained morphology (Figure 2A-D) and FACS phenotypes (Figure 2E-J) of the CLON-G treated neutrophils were not affected. The viability of the CLON-G treated neutrophils at 24 h was about 90%+ based on flow cytometry analysis (Figure 3) and the fluorescent image assays (Figure 4). Lower viability could result from improp...

Watch this Scientific Journal Video about Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay at JoVE.com

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

neutrophil-lifespan-extension-with-clon-g-an-vitro-spontaneous-death

Research

JoVE Journal

Biology

1.2K Views.  Chinese Academy of Medical Sciences & Peking Union Medical College. Ons protocol biedt een cocktailmedium om de levensduur van neutrofielen te verlengen tot meer dan vijf dagen zonder de neutrofiele functies in gevaar te brengen. Het biedt ook betrouwbare in vitro dialyse voor neutrofielen. CLON-G-behandeling breidt de mogelijkheden van neutrofielenbehoud, transport en genmanipulatie uit, wat het onderzoek in de neutrofielengemeenschap kan versnellen.Bereid om te beginnen het basismedium voor door RPMI 1640 FBS en pen-strep toe te voegen aan een buis v...

Video: Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

Genetics

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Immunology and Infection

MEDUSA for Identifying Death Regulatory Genes in Chemo-genetic Profiling Data

Systematic screening of gain- or loss-of-function genetic perturbations can be used to characterize the genetic dependencies and mechanisms of regulation for essentially any cellular process of interest. These experiments typically involve profiling from a pool of single gene perturbations and how each genetic perturbation affects the relative cell fitness. When applied in the context of drug efficacy studies, often called chemo-genetic profiling, these methods should be effective at identifying drug mechanisms of action. Unfortunately, fitness-based chemo-genetic profiling studies are ineffective at identifying all components of a drug response. For instance, these studies generally fail to identify which genes regulate drug-induced cell death. Several issues contribute to obscuring death regulation in fitness-based screens, including the confounding effects of proliferation rate variation, variation in the drug-induced coordination between growth and death, and, in some cases, the inability to separate DNA from live and dead cells. MEDUSA is an analytical method for identifying death-regulatory genes in conventional chemo-genetic profiling data. It works by using computational simulations to estimate the growth and death rates that created an observed fitness profile rather than scoring fitness itself. Effective use of the method depends on optimal tittering of experimental conditions, including the drug dose, starting population size, and length of the assay. This manuscript will describe how to set up a chemo-genetic profiling study for MEDUSA-based analysis, and we will demonstrate how to use the method to quantify death rates in chemo-genetic profiling data.

This protocol describes how to use the MEDUSA analytical method to quantify the death regulatory effect of each gene knockout. It includes instructions on determining experimental conditions that optimize sensitivity and a step-by-step tutorial on performing the analysis.

Systematic screening of gain- or loss-of-function genetic perturbations can be used to characterize the genetic dependencies and mechanisms of regulation ...

Cancer Research

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...

Neuroscience

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

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