Name: Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay (Video) | JoVE Journal
Uploaded: 2023-05-12T13:00:00.000+00:00
Duration: 5 min 52 s
Description: Learn to extend neutrophil lifespan using CLON-G medium for immunology research with mouse cells. This protocol enables reliable viability assessment through flow cytometry and confocal microscopy techniques.

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5 minutes

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

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...

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.

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

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Learn to extend neutrophil lifespan using CLON-G medium for immunology research with mouse cells. This protocol enables reliable viability assessment through flow cytometry and confocal microscopy techniques.

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

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