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 a...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;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 (CaCl2)</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>Assitant of Annexin-V binding&#160; 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&#160; 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.&#160; 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 ...

Our protocol provides a cocktail medium to prolong the neutrophil lifespan to more than five days without compromising the neutrophil functions. It also offers reliable in vitro dialysis for neutrophils. CLON-G treatment expands the possibilities of neutrophil preservation, transportation, and gene manipulation, which can accelerate research in the neutrophil community.To begin, prepare the basic medium by adding RPMI 1640 FBS and pen-strep to a 50 milliliter tube. Simultaneously, thaw all the stock solutions of the CLON-G components on ice. Then dilute HSP 70 and 200 micrograms per milliliter of G-CSF in the basic medium.Transfer 976 microliters of basic medium to a 15 milliliter centrifuge tube. To this, add the CLON-G components to prepare one milliliter of 2x CLON-G medium as described in the manuscript. Next, prepare the neutrophil culture by suspending the isolated mouse neutrophils in the basic medium.Add CLON-G medium, followed by the basic medium to 24 well non tissue cultured plates. Then add 100 microliters of the prepared cell suspension and gently pipet three to four times. Incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide supply.Stain the cultured neutrophils with cytospin and analyze them using a light microscope. For flow cytometry analysis, add one milliliter of cell medium to the neutrophil cell culture grown overnight. Mix the cell culture medium by gently pipetting it five to seven times and transfer 100 microliters of this mixture to a flow cytometry tube at the desired time points.Simultaneously, vortex the counting beads and pipet 10 microliters of bead solution to the same flow cytometry tube containing cell medium. Add 10 microliters of a pre-prepared staining mix to this flow cytometry tube and incubate for five minutes in the refrigerator under dark. Then add 100 microliters of saline to the tube and mix well for even distribution of the counting beads and cells.Finally, perform the flow cytometry analysis of the cell suspension as described in the text manuscript. For fluorescent images assay, add two milliliters of cell suspension per confocal plate and incubate at 37 degrees Celsius and 5%carbon dioxide. Meanwhile, dissolve one gram of calcium chloride in 45 milliliters of saline and filter it with a 0.2 micrometer filtering unit.Gently remove the confocal plate at the required time point. Stain the cells with 10 microliters of FITC-Annexin-V, PI and calcium chloride for five minutes at room temperature. Then using a confocal fluorescence microscope, capture the images using the desired channels.In the present study, the morphology of neutrophils was not affected in the CLON-G medium even after five days. Moreover, the fax phenotypes demonstrated intact cells after the treatment. The flow cytometry analysis showed a positive population of untreated neutrophils in Annexin-V and the population loss could be due to ethylene diamine tetraacetic acid in the cell medium.However, the viability of the CLON-G treated neutrophils at 24 hours was approximately more than 90%The fluorescent image assays also confirmed the cell viability of CLON-G treated neutrophils. The neutrophils cultured in the basic medium for 24 hours showed puffed cells and the loss of these cells might have resulted from the shaking or pipetting of the confocal plate. CLON-G compound concentrations and effects can significantly impact neutrophil tests.If at all possible, avoid modulating them. Neutrophil investigations are laborious, expensive, and extremely limited in clinical application due to their short life span. This method partially overcomes these obstacles.

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JoVE Journal

Biology

945 vistas.  Chinese Academy of Medical Sciences & Peking Union Medical College. Nuestro protocolo proporciona un medio de cóctel para prolongar la vida útil de los neutrófilos a más de cinco días sin comprometer las funciones de los neutrófilos. También ofrece diálisis in vitro confiable para neutrófilos. El tratamiento con CLON-G amplía las posibilidades de preservación, transporte y manipulación de genes de neutrófilos, lo que puede acelerar la investigación en la comunidad de neutrófilos.Para comenzar, prepare el medio básico agregando RPMI 1640...