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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 system^1,2. Granulocyte transfusion therapy has hence become a promising clinical treatment for neutropenia-related infections to transiently boost neutrophil immunity^3,4,5. 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 storage^7,8,9,10,11. 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 processes^14,15, 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 apoptosis^23,24. 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.

Protokół

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.

Wyniki

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...

Dyskusje

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. Caspase^29,<...

Materiały

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.