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 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.
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.
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.
2. In vitro spontaneous death assay of neutrophils
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: 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: 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: 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: 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.
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.
The authors have nothing to disclose.
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).
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. |