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Biology

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

Published: May 12, 2023 doi: 10.3791/65132
Automatic Translation
*1,2, *3, *4, 1,2, 5, 1,2, 5
1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 2Tianjin Institutes of Health Science, 3Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, 4Haihe Laboratory of Cell Ecosystem, Tianjin Medical University, 5Joint Program in Transfusion Medicine, Department of Pathology, Harvard Medical School; Division of Blood Bank, Department of Laboratory Medicine, The Stem Cell Program, Boston Children's Hospital, Dana-Farber /Harvard Cancer Center
* These authors contributed equally

Summary

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.

Abstract

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.

Introduction

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 µM 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.

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Protocol

The Boston Children'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.

  1. Preservation of the CLON-G components
    1. Dissolve 50 mg of Q-VD-oph (see Table of Materials) to a final concentration of 100 mM by adding 973.7 µL of dimethyl sulfoxide (DMSO), and pipette the liquid until the mixture becomes clear. Aliquot 50 µL per tube, and preserve at −20 °C.
      CAUTION: DMSO is a harmful chemical liquid. Wear a lab coat, goggles, and a mask to avoid skin contact, eye contact, and inhalation.
    2. Thaw Hsp70 (see Table of Materials) at 4 °C, and spin down the contents in the vial. Aliquot 1 µL of Hsp70 per tube on ice, and preserve at −80 °C.
      NOTE: Hsp70 is a protein; ultra-cold storage is necessary to keep it stable. Avoid the freeze-thaw cycle.
    3. Dissolve 1 mg of DFO (see Table of Materials) with 7.61 mL RPMI 1640 to obtain a stock of 200 µM. Aliquot 500 µL per tube, and preserve at −20 °C.
    4. 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 µm filtering unit. Aliquot 500 µL per tube, and preserve at −20 °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 −20 °C.
    5. 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 µL per tube, and preserve at −20 °C. Protect from light.
    6. Dissolve 250 µg of G-CSF (see Table of Materials) to 200 µg/mL with 1.25 mL of RPMI 1640. Aliquot 50 µL per tube, and preserve at 4 °C.
  2. Preparation of 2x CLON-G culture medium
    1. 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.
    2. Thaw all the stock solutions of the CLON-G components on ice.
    3. Dilute 1 µL of Hsp70 to 20 µM by adding 606 µL of the basic medium. Dilute 1 µL of 200 µg/mL G-CSF to 20 µg/mL by adding 9 µL of the basic medium.
    4. Add 976 µL of basic medium to a 15 mL centrifuge tube. Add 1 µL of Q-VD-oph (100 mM), 10 µL of DFO (200 µM), 1 µL of Hsp70 (20 µM), 10 µL of NAC (200 mM), 1 µL of Nec-1s (20 mM), and 1 µL of G-CSF (20 µg / 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 °C. Leftover 20 µM Hsp70 must be discarded.
  3. Neutrophil culture
    1. Isolate mouse neutrophils by gradient centrifugation following a previous report25. Resuspend the isolated mouse neutrophils in the basic medium (1 million cells/100 µL).
      NOTE: Avoid activating the neutrophils by handling them gently. Avoid foaming during the whole process.
    2. Add 500 µL of 2x CLON-G medium, 400 µL of the basic medium, and 100 µL 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.
    3. Place the culture plate in a 37 °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 µM Q-VD-Oph, 1 µM DFO, 10 pM Hsp70, 1 mM NAC, 10 µM Nec-1 s, and 10 ng/mL G-CSF.
    4. 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.

2. In vitro spontaneous death assay of neutrophils

  1. Flow cytometry analysis
    1. Culture isolated neutrophils at a density of 1 million cells/mL in the basic medium at 37 °C and 5% CO2. The 24-well culture plate is non-tissue cultured. Add 1 mL of cell medium per well.
    2. Dissolve 1 g of CaCl2 with 45 mL of saline to make 200 mM CaCl2. Filter it with a 0.2 µm filtering unit. Preserve at 4 °C.
    3. Prepare the staining mix. Add 7 µL 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 µL each per sample. Mix well. Use the solution immediately after preparation.
    4. Pipette the cell medium gently five to seven times to mix well, followed by transferring 100 µL to a flow cytometry tube at the desired time points.
      NOTE: Avoid foaming during the whole process.
    5. Vortex counting beads (see Table of Materials) to mix them well. Pipette 10 µL of the well-mixed counting beads to the same flow cytometry tube as in step 2.1.4.
    6. Add 10 µL the prepared staining mix (step 2.1.3) to the flow cytometry tube. Incubate at room temperature for 5 min away from light.
    7. Add 100 µL of saline to the tube, and mix well to ensure the even distribution of the counting beads and cells.
    8. 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−PI− 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) × number of Annexin-V−PI− population × 10
      ​Viability of neutrophils = total number of healthy cells at the indicated time/total number of healthy cells at time zero
  2. Fluorescent image assay
    1. Culture isolated neutrophils at a density of 1 million cells/mL in the basic medium at 37 °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.
    2. Take the plate out gently at the indicated time point. Add 10 µL 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.
    3. 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.

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Representative Results

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 improper storage, improper concentrations of the CLON-G components, or poor quality of the starting isolated neutrophils. The flow cytometry analysis of the untreated neutrophils that were cultured with the basic medium for 24 h showed that these neutrophils had an Annexin-V-positive population (Figure 3B). The loss of this population could be due to ethylene diamine tetraacetic acid (EDTA) in the cell medium. The fluorescence images of the neutrophils cultured with the basic medium for 24 h should contain puffed cells (white arrow in Figure 4A). The loss of puffed cells might result from the shaking or pipetting of the confocal plate.

Figure 1
Figure 1: Flow chart of neutrophil culturing with CLON-G and the in vitro death assay. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Morphology and cell surface marker phenotypes of the CLON-G-treated mouse neutrophils. The cells were (A-D) stained with Wright-Giemsa compound stain after culturing and assessed by microscopy using a 40x objective lens or (E-I) stained with APC-CD11b and PE-Cy7-Ly6G antibodies and analyzed with flow cytometry. (J) The ratios of the CD11b+ and Ly6G+ cells at the indicated time points were statistically analyzed. The scale bar is 10 µm. Data are presented as means ± SD of three experiments. ns = no statistically significant difference compared to the corresponding group. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Flow cytometry analysis of CLON-G-treated mouse bone marrow neutrophils. (A) Gating strategy, (B) representative results, and (C) the viability of the neutrophils after culturing for 24 h. Data are presented as means ± SD of three experiments. **P < 0.001 compared to the corresponding group. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Representative results for neutrophil death based on the fluorescence image assay. Neutrophil death after culturing with (A) basic medium for 24 h or CLON-G for (B) 24 h, (C) 3 days, or (D) 5 days. After culturing, the cells were stained with FITC-Annexin-V (Green) and PI (Red) and assessed by confocal fluorescence microscopy. The scale bar is 40 µm. Please click here to view a larger version of this figure.

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Discussion

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. Caspase29,30,31,32, LMP33, ROS21,22,33, necrosis34,35,36,37, and necropotosis18,38 have been proven to be involved in these heterogeneous processes. GM-CSF24and G-CSF23 can prolong the neutrophil lifespan to about 24 h with suppressed functions. CLON-G targets all known neutrophil spontaneous death mechanisms. To the best of our knowledge, CLON-G currently provides the longest elongation of the neutrophil life span in vitro without compromising function.

Several caveats must be considered to obtain the best results from neutrophil culturing with CLON-G. Through our previous pharmacological screening16, it was emphasized that accurate concentrations of the CLON-G components are key to success and, thus, that one should try to avoid making any mistakes during the preparation and to preserve them properly. Regarding the neutrophil manipulation and purification, it is easy to activate and kill the neutrophils; therefore, they should be treated gently during the whole process and cultured once they are isolated. They are sensitive to the cell concentration, the condition of the culture plate, temperature, and pH; one must be careful when changing this protocol. With regard to the in vitro death assay, previous results have demonstratedthat spontaneously dead neutrophils swell to puffed cells that are intolerable of mechanical force; spinning down, pipetting, and washing would reduce the number of neutrophils, and replacing the standard binding buffer with a CaCl2 solution can simplify this process and ensure the accurate depiction of the neutrophils14. Discrepancies among the technical duplicates in the same experiment could result from foaming during the operation. Differences among independent experiments might be due to variations in the purity and freshness of the neutrophils39.

CLON-G combines known pathways and corresponding inhibitory chemicals to prevent spontaneous neutrophil death. However, the core question of this field remains regarding the complicated pathways that mediate neutrophil death, as these are still not fully understood. The death of CLON-G-treated neutrophils is inevitable. Thus, CLON-G still has room for improvement. As for the components of CLON-G, the best choices are clinically applied drugs to expand the possibility of the clinical application of CLON-G-treated neutrophils; however, Q-VD-oph and Rec-1s are for research only, and clinical trials focusing on them are currently unavailable. Thus, alternative compounds are necessary for clinical application.

By prolonging the neutrophil lifespan to greater than 5 days with their functions intact, CLON-G can unlock endless possibilities for neutrophil research. With this extended window, observation, gene manipulation, long-term storage, transportation, and cryopreservation can be developed based on CLON-G. The labor, time, and money costs would be reduced substantially, which would lower the entry barriers for new researchers. CLON-G can also promote neutrophil clinical applications like granulocyte transfusion therapy. Neutropenic infection post-chemotherapy is a major cause of death for patients who suffer from cancer and hematopoietic malignancies40,41,42,43,44. Granulocyte transfusion therapy, which has great potential as an alternative therapy to aid these patients, is severely limited by the short lifespan of neutrophils45. The current process flow for granulocyte transfusion takes longer than the lifespan of a neutrophil, leading to transfused neutrophils losing their effectiveness as a first-line immune cell. CLON-G treatment provides enough time for the preparation of granulocyte transfusions, which have the potential to save countless lives of neutropenic-infected patients and to help mitigate the overuse of antibiotics. Concurrently, the present protocol for the neutrophil death assay can accurately demonstrate the status of neutrophils and improve the replicability of experimental results.

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Disclosures

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

Acknowledgments

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-1-040，2022-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).

Materials

Name Company Catalog Number Comments
0.2 µm syringe filter Pall Corporation 4612 Filtrate prepared CLON-G components.
1.5 mL micro centrifuge tube LABSELECT MCT-001-150 Lab consumable.
15 mL Centrifuge Tubes LABSELECT CT-002-15 Lab consumable.
24 well cell culture plate Falcon 351147 Neutrophil culture plate.
50 mL Centrifuge Tubes LABSELECT CT-002-50 Lab consumable.
BD LSRII BD Instrument for flow cytometry analysis of neutrophil death.
Calcium chloride (CaCl2) Sigma Aldrich C4901 Assitant of Annexin-V binding  to phosphatidylserine.
Confocal microscope Perkinelmer UltraVIEW VOX Instrument for fluorescent analysis of neutrophil death.
Confocal plate NEST 801001-20mm Lab consumable for fluorescent image assay.
Counting beads Thermo Fisher C36950 Quantification in flow cytometry analysis of neutrophil death.
DFO Sigma Aldrich D9533 Component of CLON-G. LMP inhibitor.
Dimethyl sulfoxide ( DMSO) Sigma Aldrich D2650 Solvent for Q-VD-oph and Nec-1s.
Fetal Bovine Serum Gibco 10099141C Component of neutrophil culture basic medium. Nutrition supply.
FITC-Annexin-V BD 51-65874X Annexin-V can bind to phosphatidylserine of aged cells.This is at FITC channel.
Hsp70 Abcam ab113187 Component of CLON-G. LMP inhibitor.
NAC Sigma Aldrich A9165 Component of CLON-G. Antioxidant.
Nec-1s EMD Millipore 852391-15-2 Component of CLON-G. Necroptosis inhibitor.
Penicillin-Streptomycin Solution (PS) Gibco 15070063 Component of neutrophil culture basic medium. Antibiotics to protect cells from bacteria comtamination.
Propidium Iodide (PI) BioLegend 421301 For neutrophil death assay. A small fluorescent molecule that binds to DNA  but cannot passively traverse into cells that possess an intact plasma membrane.
Q-VD-oph Selleck chem S7311 Component of CLON-G. Pan-caspase inhibitor.
Recombinant Human Granulocyte Colony-stimulating Factor for Injection (CHO cell)(G-CSF) Chugai Pharma China GRANOCYTE Component of CLON-G.  Promote neutrophil survival through Akt pathway.
Round-Bottom Polystyrene Tubes Falcon 100-0102 Lab consumable for flow cytometry analysis.
RPMI1640 Gibco C11875500BT Component of neutrophil culture basic medium.
Saline LEAGENE R00641 Solution for flow cytometry analysis of neutrophil death.
Sodium hydroxide (NaOH) FENG CHUAN 13-011-00029 pH adjustion for NAC.
Wright-Giemsa Stain Solution Solarbio G1020 Neutrophil cytospin staining.

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References

  1. Segal, A. W. How neutrophils kill microbes. Annual Review of Immunology. 23, 197-223 (2005).
  2. Rosales, C. Neutrophil: A cell with many roles in inflammation or several cell types. Frontiers in Physiology. 9, 113 (2018).
  3. Chung, S., Armstrong-Scott, O., Charlewood, R. Therapeutic granulocyte infusion for patients with severe neutropaenia and neutrophilic dysfunction: New Zealand experience. Vox Sanguinis. 117 (2), 220-226 (2021).
  4. Zhou, B., et al. Clinical outcome of granulocyte transfusion therapy for the treatment of refractory infection in neutropenic patients with hematological diseases. Annals of Hematology. 97 (11), 2061-2070 (2018).
  5. Covas, D. T., et al. Granulocyte transfusion combined with granulocyte colony stimulating factor in severe infection patients with severe aplastic anemia: A single center experience from China. PLoS One. 9 (2), e88148 (2014).
  6. Tecchio, C., Cassatella, M. A. Uncovering the multifaceted roles played by neutrophils in allogeneic hematopoietic stem cell transplantation. Cellular & Molecular Immunology. 18 (4), 905-918 (2021).
  7. Lahoz-Beneytez, J., et al. Human neutrophil kinetics: Modeling of stable isotope labeling data supports short blood neutrophil half-lives. Blood. 127 (26), 3431-3438 (2016).
  8. Pillay, J., et al. In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood. 116 (4), 625-627 (2010).
  9. Kolaczkowska, E., Kubes, P. Neutrophil recruitment and function in health and inflammation. Nature Reviews Immunology. 13 (3), 159-175 (2013).
  10. Tofts, P. S., Chevassut, T., Cutajar, M., Dowell, N. G., Peters, A. M. Doubts concerning the recently reported human neutrophil lifespan of 5.4 days. Blood. 117 (22), 6050-6052 (2011).
  11. McDonald, J. U., et al. In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils. FASEB Journal. 25 (6), 1972-1982 (2011).
  12. Pedruzzi, E., Fay, M., Elbim, C., Gaudry, M., Gougerot-Pocidalo, M. -A. Differentiation of PLB-985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N-formyl peptide and priming of superoxide anion production by granulocyte-macrophage colony-stimulating factor. British Journal of Haematology. 117 (3), 719-726 (2002).
  13. Blanter, M., Gouwy, M., Struyf, S. Studying neutrophil function in vitro: Cell models and environmental factors. Journal of Inflammation Research. 14, 141-162 (2021).
  14. Teng, Y., Luo, H. R., Kambara, H. Heterogeneity of neutrophil spontaneous death. American Journal of Hematology. 92 (8), E156-E159 (2017).
  15. Luo, H. R., Loison, F. Constitutive neutrophil apoptosis: Mechanisms and regulation. American Journal of Hematology. 83 (4), 288-295 (2008).
  16. Fan, Y., et al. Targeting multiple cell death pathways extends the shelf life and preserves the function of human and mouse neutrophils for transfusion. Science Translational Medicine. 13 (604), (2021).
  17. Caserta, T. M. Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis. 8, 345-352 (2003).
  18. Wang, X., Yousefi, S., Simon, H. U. Necroptosis and neutrophil-associated disorders. Cell Death and Disorders. 9 (2), 111 (2018).
  19. Loison, F., et al. Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation. Journal of Clinical Investigation. 124 (10), 4445-4458 (2014).
  20. Kambara, H., et al. Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death. Cell Reports. 22 (11), 2924-2936 (2018).
  21. Zhu, D., et al. Deactivation of phosphatidylinositol 3,4,5-trisphosphate/Akt signaling mediates neutrophil spontaneous death. Proceedings of the National Academy of Sciences of the United States of America. 103 (40), 14836-14841 (2006).
  22. Xu, Y., Loison, F., Luo, H. R. Neutrophil spontaneous death is mediated by down-regulation of autocrine signaling through GPCR, PI3Kgamma, ROS, and actin. Proceedings of the National Academy of Sciences of the United States of America. 107 (7), 2950-2955 (2010).
  23. van Raam, B. J., Drewniak, A., Groenewold, V., vanden Berg, T. K., Kuijpers, T. W. Granulocyte colony-stimulating factor delays neutrophil apoptosis by inhibition of calpains upstream of caspase-3. Blood. 112 (5), 2046-2054 (2008).
  24. Klein, J. B., et al. Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. Journal of Immunology. 164 (8), 4286-4291 (2000).
  25. Fan, Y., et al. Targeting multiple cell death pathways extends the shelf life and preserves the function of human and mouse neutrophils for transfusion. Science Translational Medicine. 13 (604), (2021).
  26. Xie, X., et al. Single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection. Nature Immunology. 21 (9), 1119-1133 (2020).
  27. Cosimo, E., et al. Inhibition of NF-kappaB-mediated signaling by the cyclin-dependent kinase inhibitor CR8 overcomes prosurvival stimuli to induce apoptosis in chronic lymphocytic leukemia cells. Clinical Cancer Research. 19 (9), 2393-2405 (2013).
  28. Borregaard, N. Neutrophils, from marrow to microbes. Immunity. 33 (5), 657-670 (2010).
  29. Porter, A. G., Jänicke, R. U. Emerging roles of caspase-3 in apoptosis. Cell Death and Differentiation. 6 (2), 99-104 (1999).
  30. Scheel-Toellner, D., et al. Clustering of death receptors in lipid rafts initiates neutrophil spontaneous apoptosis. Biochemical Society Transactions. 32, 679-681 (2004).
  31. Geering, B., Simon, H. U. Peculiarities of cell death mechanisms in neutrophils. Cell Death and Differentiation. 18 (9), 1457-1469 (2011).
  32. Gabelloni, M. L., Trevani, A. S., Sabatté, J., Geffner, J. Mechanisms regulating neutrophil survival and cell death. Seminars in Immunopathology. 35 (4), 423-437 (2013).
  33. Loison, F., et al. Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation. Journal of Clinical Investigation. 124 (10), 4445-4458 (2014).
  34. Cho, Y. S., et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 137 (6), 1112-1123 (2009).
  35. He, S., et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 137 (6), 1100-1111 (2009).
  36. Sun, L., et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 148 (1-2), 213-227 (2012).
  37. Zhang, D. -W., et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science. 325 (5938), 332-336 (2009).
  38. Wang, X., He, Z., Liu, H., Yousefi, S., Simon, H. -U. Neutrophil necroptosis is triggered by ligation of adhesion molecules following GM-CSF priming. Journal of Immunology. 197 (10), 4090-4100 (2016).
  39. Hsu, A. Y., Peng, Z., Luo, H., Loison, F. Isolation of human neutrophils from whole blood and buffy coats. Journal of Visualized Experiments. (175), e62837 (2021).
  40. George, B., Bhattacharya, S. Infections in hematopoietic stem cell transplantation (HSCT) patients. Contemporary Bone Marrow Transplantation. Chandy, M., Radhakrishnan, V. S., Sukumaran, R. , Springer. Cham, Switzerland. (2020).
  41. Lucena, C. M., et al. Pulmonary complications in hematopoietic SCT: A prospective study. Bone Marrow Transplant. 49 (10), 1293-1299 (2014).
  42. Tomblyn, M., et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: A global perspective. Biology of Blood and Marrow Transplantation. 15 (10), 1143-1238 (2009).
  43. Wingard, J. R., Hiemenz, J. W., Jantz, M. A. How I manage pulmonary nodular lesions and nodular infiltrates in patients with hematologic malignancies or undergoing hematopoietic cell transplantation. Blood. 120 (9), 1791-1800 (2012).
  44. Wingard, J. R., Hsu, J., Hiemenz, J. W. Hematopoietic stem cell transplantation: An overview of infection risks and epidemiology. Infectious Disease Clinics of North America. 24 (2), 257-272 (2010).
  45. Netelenbos, T., et al. The burden of invasive infections in neutropenic patients: incidence, outcomes, and use of granulocyte transfusions. Transfusion. 59 (1), 160-168 (2019).

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Neutrophil Lifespan Extension CLON-G In Vitro Spontaneous Death Assay Cocktail Medium Neutrophil Functions Dialysis Preservation Transportation Gene Manipulation RPMI 1640 FBS Pen-strep HSP 70 G-CSF Centrifuge Tube Neutrophil Culture Non Tissue Cultured Plates Cell Suspension Incubate Stain Cytospin Light Microscope Flow Cytometry Analysis
Neutrophil Lifespan Extension with CLON-G and an <em>In Vitro</em> Spontaneous Death Assay
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Fan, Y., Teng, Y., Liu, F. t., Ma,… More

Fan, Y., Teng, Y., Liu, F. t., Ma, F., Hsu, A. Y., Feng, S., Luo, H. R. Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay. J. Vis. Exp. (195), e65132, doi:10.3791/65132 (2023).

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