Method for inducing HL-60 cells to generate neutrophil extracellular trap net and application thereof

Abstract

The invention discloses a method for inducing HL-60 cells to generate a neutrophil extracellular trap net and application of the method. The method comprises the following steps: firstly, inducing and differentiating HL-60 cells into neutrophile granulocyte-like cells by using dimethyl sulfoxide (DMSO), and then stimulating the differentiated cells by using phorbol ester (PMA), thereby inducing to form the neutrophile granulocyte extracellular trapping net. Preferably, the final concentration of the DMSO is 0.8%-1.5%, the final concentration of the PMA is 50-400 nM, and the stimulation time is 2-4 hours. According to the invention, a polylysine-coated substrate is adopted to enhance the structural stability of NETs, and SYTOX Green nucleic acid dye is adopted for fluorescence detection. According to the invention, a complete and standardized experimental system formed from the HL-60 cell to the NETs is successfully established, the technical problems of unstable primary cell source and poor experimental repeatability are overcome, the operation is simple and convenient, the result is reliable, and an ideal tool is provided for related research of the NETs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for inducing HL-60 cells to produce extracellular neutrophil traps (NETs) and its application. Background Technology

[0002] Neutrophil extracellular traps (NETs) are fibrous network structures composed of a DNA backbone and various granular proteins released extracellularly by neutrophils upon activation. NETs can encapsulate and fix microorganisms such as bacteria, fungi, and viruses, playing an important role in innate immune responses, infection clearance, thrombosis, and autoimmune diseases. The formation mechanism of NETs involves the production of reactive oxygen species, nuclear degeneration, and chromatin efflux. After release, the fibrous expansion signal can be observed under fluorescent dye labeling. Because the formation of NETs is strictly dependent on cell state, constructing a reproducible and operable in vitro induction and detection system is crucial for related basic research and drug screening.

[0003] Traditional research often relies on inducing NETs from primary neutrophils isolated from human peripheral blood. Typical methods include obtaining cells through density gradient centrifugation, induction with stimulants such as phorbol ester (PMA), and observation via fluorescent staining. However, the source of primary neutrophils is limited, the acquisition process is cumbersome, and it is significantly affected by individual donor differences. More importantly, neutrophils have a very short survival time in vitro and their activity decays rapidly, resulting in poor reproducibility and stability of NETs induction experiments, making it difficult to meet the needs of standardized research.

[0004] To circumvent the defects of primary neutrophils, researchers have gradually attempted to use cell lines with myeloid differentiation capabilities to replace primary cells, among which the human promyelocytic leukemia cell line HL-60 is the most widely used model. Existing techniques show that treating HL-60 cells with inducing agents such as dimethyl sulfoxide (DMSO) or retinoic acid (ATRA) for 4-6 days can induce a neutrophil-like phenotype, characterized by nuclear segmentation, upregulated expression of the CD11b surface marker, and enhanced phagocytic capacity. Patent document CN202511218028 also discloses a method for optimizing HL-60 cell differentiation efficiency through the combination of DMSO and retinoic acid. However, these existing techniques focus entirely on the "cell differentiation" stage itself, without addressing or providing any indication of how to further induce differentiated cells to form NETs. Because differentiation capacity alone is insufficient to guarantee NET formation, undifferentiated or poorly differentiated HL-60 cells, even when chemically stimulated, primarily release extracellular DNA as unstructured fragments or cell death products, failing to form NETs with typical extended characteristics. Therefore, it is necessary to further induce NET formation after successful HL-60 differentiation using appropriate stimulation methods. However, a complete and readily applicable method for this process is still lacking in existing experimental systems.

[0005] In summary, the current research status presents a technological gap: on the one hand, NETs induction methods based on primary cells, while effective, are unstable; on the other hand, HL-60 cell-based approaches only address the differentiation problem but fail to establish the functional induction pathway to NET formation. This gap has resulted in a long-standing lack of a standardized technical approach in the field that can continuously and reliably induce and observe NETs from readily available and stable cell sources. This invention aims to fill this technological gap. Summary of the Invention

[0006] The present invention aims to address the lack of a complete and standardized method in the prior art for continuously and reliably inducing the formation of extracellular neutrophil traps (NETs) from a stable cell source, and to provide a simple, stable and reproducible method for inducing NETs using HL-60 cells.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for inducing HL-60 cells to generate an extracellular trap for neutrophils.

[0008] The method includes the following steps: Provide HL-60 cells; The HL-60 cells were induced to differentiate into neutrophil-like cells using dimethyl sulfoxide; The neutrophil-like cells were stimulated with phorbol ester to induce the formation of extracellular neutrophil traps.

[0009] The core of the above method lies in achieving efficient NET formation on the HL-60 cell line through a continuous and optimized two-step process.

[0010] Preferably, in the differentiation step, the final concentration of dimethyl sulfoxide (DMSO) is 0.8% to 1.5% (v / v). More preferably, the final concentration of DMSO is 1.25% (v / v). DMSO, as a chemical inducer, functions to initiate the differentiation program of HL-60 cells toward neutrophils, enabling them to acquire a neutrophil-like phenotype.

[0011] Preferably, in the stimulation step, the final concentration of phorbol ester (PMA) is from 50 nM to 400 nM. More preferably, the final concentration of PMA is 100 nM. PMA, as a potent activator of protein kinase C (PKC), can specifically activate the NETosis signaling pathway.

[0012] Preferably, the stimulation time with phorbol ester (PMA) is 2 to 4 hours. More preferably, the stimulation time is 3 to 4 hours, which is a critical period for observing the formation of a large number of mature NETs structures.

[0013] Preferably, the step of inducing the formation of neutrophil extracellular traps is performed on a substrate coated with poly-L-lysine. Poly-L-lysine is a positively charged amino acid polymer that can significantly enhance the adhesion of cells and NETs DNA structures to the substrate through electrostatic adsorption, effectively preventing the structures from detaching during washing.

[0014] Preferably, SYTOX Green nucleic acid dye is used to fluorescently label and observe the formed extracellular traps of neutrophils. SYTOX Green is a nucleic acid dye that cannot penetrate the membrane of living cells, thus it can specifically label extracellular DNA, achieving high signal-to-noise ratio fluorescence observation without the need for complex immunofluorescence techniques.

[0015] A second aspect of the present invention provides the application of the method described in the first aspect of the present invention in the preparation of reagents or models for drug screening, research on inflammation mechanisms, or construction of disease models.

[0016] The beneficial effects of this invention are as follows: This invention establishes for the first time a complete, continuous, and standardized induction system from HL-60 cell source to NET functional realization, successfully overcoming the core challenges of existing technologies that rely on primary neutrophils, resulting in limited cell source, significant individual variability, and poor experimental reproducibility. By clarifying the key steps and optimizing parameters for DMSO differentiation and PMA induction (e.g., preferably 1.25% DMSO and 100 nM PMA), the NET formation process becomes highly controllable and the results predictable, significantly improving the success rate and reliability of the experiment. Crucially, the introduction of a poly-L-lysine-coated substrate effectively enhances the stability of the fragile NET structure, while the use of SYTOX Green for single-channel fluorescence detection greatly simplifies the observation process and reduces reliance on complex equipment and operational skills. Ultimately, this invention provides a powerful, stable, and reproducible tool for basic NET mechanism research, drug screening, and disease model construction, possessing significant scientific research value and application prospects. Attached Figure Description

[0017] Figure 1 The figure shows the flow cytometry results of HL-60 cells induced by different concentrations of DMSO in Example 1 of this invention. The figure shows the detection results of the blank control group (A) and the treatment groups with different final concentrations of DMSO (B: 0.8%, C: 1.0%, D: 1.25%, E: 1.5%).

[0018] Figure 2 This is a fluorescence micrograph of neutrophil-like cells (HL-60 cells induced to differentiate by 1.25% DMSO) obtained in Example 1 by stimulating different concentrations of PMA in Example 2 of the present invention. The figure shows the cell morphology after treatment with 50 nM, 100 nM and 400 nM PMA at a fixed stimulation time.

[0019] Figure 3 The image shows fluorescence micrographs of neutrophil-like cells (HL-60 cells induced to differentiate by 1.25% DMSO) obtained in Example 1 by PMA stimulation at different times in Example 2 of the present invention. The image shows the cell morphology after stimulation for 1 hour, 2 hours and 4 hours at a fixed PMA concentration.

[0020] Figure 4 This is a fluorescence micrograph comparing HL-60 cells under different experimental conditions in Example 2 of the present invention. The figure shows the cell morphology of HL-60 cells under three treatment conditions: HL-60 cells without DMSO induction and PMA stimulation, HL-60 cells induced by DMSO without PMA stimulation, and HL-60 cells induced by DMSO and PMA stimulation (magnification: 10×, 20×, 40×). Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All materials and instruments used in the embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Example 1: Induction of HL-60 cells to differentiate into neutrophil-like cells This embodiment aims to provide a specific method for efficiently inducing HL-60 cells to differentiate into neutrophil-like cells, and to determine the optimal concentration of the inducing agent through experiments, so as to provide well-prepared cell material for the subsequent induction of extracellular neutrophil traps (NETs).

[0023] The materials and instruments used in this embodiment include: HL-60 cell line (ATCC CCL-240), IMDM medium (Gibco), fetal bovine serum (FBS, Gibco), dimethyl sulfoxide (DMSO, analytical grade), PBS buffer (pH 7.4), CD11b-FITC antibody (Biolegend), and flow cytometer (BD FACSCanto II).

[0024] The specific experimental steps in this embodiment are as follows: (1) Cell resuscitation and passage: Remove the HL-60 cryovials from liquid nitrogen, thaw them in a 37°C water bath, centrifuge to remove the cryopreservation solution, and resuspend them in IMDM complete medium containing 20% ​​FBS. Incubate the cells in a 37°C, 5% CO2 incubator until the cells are in good growth condition and enter the logarithmic growth phase (density approximately 1×10⁻⁶). 6 Experiments were conducted when the cell count was >95% (cells / mL).

[0025] (2) Experimental grouping: To determine the optimal induction conditions, different concentrations of DMSO were used in the experiment. The cell density was adjusted to 3 × 10⁶ cells / year. 5 Cells / mL were seeded in T-25 culture flasks, and IMDM complete medium was used as the basal medium. The experiment was divided into the following five groups: blank control group (no DMSO), low concentration group (final concentration 0.8% DMSO), medium concentration group (final concentration 1.0% DMSO), high concentration group 1 (final concentration 1.25% DMSO), and high concentration group 2 (final concentration 1.5% DMSO).

[0026] (3) Induction culture: The cells in each group were placed in a 37℃, 5% CO2 incubator and cultured continuously for 4 days (96 hours). During this period, half of the medium was replaced every 48 hours, and fresh medium containing the corresponding concentration of DMSO was added to maintain a constant concentration of inducer.

[0027] (4) Flow cytometry: After culture, collect cells from each group and wash twice with PBS. Each 1×10⁶ cells... 6 Each cell was resuspended in 100 μL of PBS, and 5 μL of FITC-labeled anti-human CD11b antibody was added. The cells were incubated in the dark for 20 minutes. After incubation, the cells were washed twice with PBS, resuspended in 400 μL of PBS, and immediately analyzed by flow cytometry. Differentiation efficiency was assessed by analyzing the proportion of CD11b-positive cells.

[0028] Flow cytometry results as follows Figure 1 As shown in Table 1, compared with the blank control group (CD11b positivity rate <10%), DMSO treatment significantly induced HL-60 cells to differentiate into neutrophil-like cells. The CD11b positivity rate increased with increasing DMSO concentration, reaching a peak of 89.4% at a concentration of 1.25%. When the concentration increased to 1.5%, the positivity rate decreased slightly. Microscopic observation revealed typical morphological changes in the cells of the 1.25% DMSO group, such as a decreased nucleus-to-cytoplasm ratio and the appearance of segmented nuclei, and the cells were in good condition. Considering both differentiation efficiency and cell viability, this invention determined that 1.25% is the optimal concentration of DMSO for inducing HL-60 cells to differentiate into neutrophil-like cells.

[0029] Table 1. CD11b positivity rate of HL-60 cells induced by different concentrations of DMSO for 4 days (n=3)

[0030] Example 2: NETs Induction and Optimization Observation This embodiment aims to verify that the neutrophil-like cells obtained by DMSO-induced differentiation of HL-60 cells in Example 1 can indeed produce typical NETs structures after further induction, and to optimize key induction parameters and observation conditions to establish a stable and reproducible NETs induction and detection system.

[0031] The materials and instruments used in this embodiment include: neutrophil-like cells induced to differentiate by 1.25% DMSO in Example 1, serum-free IMDM medium, phorbol ester (PMA, Sigma-Aldrich, catalog number P1585, prepared as a 1 mM stock solution using DMSO, aliquoted and stored at -20°C), SYTOX Green nucleic acid dye (Thermo Fisher Scientific, catalog number S7020), phosphate-free buffer (such as HBSS or Tris-HCl, used for washing and dilution to avoid the influence of phosphate in PBS on staining efficiency), 24-well glass plates (pre-coated with 0.01% poly-L-lysine (Sigma-Aldrich, catalog number P4707) to enhance the physical adhesion of cells and DNA structures), and a fluorescence microscope (equipped with a FITC filter).

[0032] The specific experimental steps in this embodiment are as follows (PMA concentration optimization): (1) NETs induction: Neutrophil-like cells obtained by HL-60 cells induced by 1.25% DMSO in Example 1 were collected, resuspended in serum-free IMDM medium, and the cell density was adjusted to 2×10⁻⁶. 5 Cells / mL. 1 mL of cell suspension was seeded into a 24-well plate coated with poly-L-Lysine. Then, different concentrations of PMA (final concentrations of 50 nM, 100 nM, and 400 nM) were added to the wells, and the cell culture plate was incubated at 37°C in a 5% CO2 incubator for 3–4 hours.

[0033] Unlike traditional NETs induction experiments, this invention keyly employs a poly-L-lysine-coated substrate to solve the technical problem of NETs being fragile and easily detached during washing, thereby significantly improving the retention rate of the structure and the clarity of observation.

[0034] (2) Fluorescent Staining and Observation: After incubation, carefully aspirate the supernatant to avoid disturbing the cells and NETs structure at the bottom. Slowly add pre-warmed HBSS buffer along the well wall and wash gently twice. Then, add 300 μL of HBSS working solution containing 167 nM SYTOX™ Green dye to each well, ensuring complete coverage of the bottom. Incubate at room temperature in the dark for 15 minutes. After incubation, aspirate the staining solution and wash gently twice again with HBSS buffer to remove unbound dye. Finally, add a small amount of HBSS to keep the well moist, and immediately observe and acquire images using the FITC channel under a fluorescence microscope.

[0035] Surprisingly, we found that using only SYTOX Green for single-channel staining could yield clear NETs images with low background and high signal-to-noise ratio. This contrasts sharply with existing technologies that typically require complex immunofluorescence colocalization (such as MPO / DNA or NE / DNA colabeling) to confirm NETs, ​​greatly simplifying the detection process and lowering the technical threshold and equipment requirements.

[0036] (3) Condition optimization - stimulus time In steps (1) and (2) above, other conditions remained unchanged, the PMA concentration was adjusted to 100 nM, and the incubation time was set to 1 hour (1H), 2 hours (2H), and 4 hours (4H) respectively. The effects of different stimulation times on NETs formation were observed.

[0037] (4) Negative control verification To verify the specificity of NET formation, the following negative control and experimental groups were set up: Control 1: HL-60 cells were not induced by DMSO (undifferentiated cells) and were stimulated with PMA; Control 2: HL-60 cells were induced with DMSO (differentiated cells) without the addition of PMA stimulation; Experimental group: HL-60 cells were induced by DMSO (referred to as differentiated cells) and then stimulated with 100 nM PMA.

[0038] The optimized PMA concentration results are as follows: Figure 2 As shown, different PMA concentrations significantly affect NET formation. Stimulation with 50 nM PMA for 4 hours only induces a small number of sparse NET structures; stimulation with 100 nM PMA for 4 hours induces a large number of typical, well-extended filamentous and network NET structures; while 400 nM PMA leads to excessive cell activation and death, producing a large amount of non-specific DNA fragments and unclear network structures. Therefore, 100 nM was determined to be the optimal PMA concentration for inducing NET formation.

[0039] Stimulation time optimization results are as follows Figure 3 As shown, the formation process of NETs was observed at different time points under 100 nM PMA stimulation: after 1 hour of stimulation, the cell nuclei began to depolymerize, but no obvious extracellular structures were observed; after 2 hours of stimulation, some cells began to release DNA, forming initial filaments; after 3 to 4 hours of stimulation, a large number of mature, interconnected NET structures were formed, with the most typical and complete morphology; after 4 hours, signs of degradation began to appear. Therefore, 3-4 hours was determined to be the optimal observation window.

[0040] Induced necessary condition verification and NETs morphological results, such as Figure 4As shown, in the negative controls 1 (undifferentiated cells + PMA) and 2 (differentiated cells, without PMA), only intact, round nuclear fluorescence was observed in the field of view, without any extracellular filamentous or reticular structures. In stark contrast, in the experimental group (differentiated cells + PMA), under optimal conditions (100 nM PMA, 3-4 h), the release of SYTOX™ Green-labeled green fluorescence signals from the cell nucleus was clearly observed, forming typical cloud-like, filamentous, and reticular morphologies. The structure extended significantly beyond the cell body and formed complex connections between multiple cells. This result confirms that "DMSO-induced differentiation" and "PMA stimulation" are two indispensable conditions for HL-60 cells to produce NETs. The NETs positive criteria established in this invention are: (1) the appearance of continuous extracellular filamentous or network DNA fluorescent structures; (2) the structure clearly breaks through the cell nuclear boundary; and (3) excluding dead cell fragments that only show punctate fluorescence.

[0041] The key feature of this invention is the use of poly-L-lysine-coated substrates, which effectively enhances the adhesion of cells and fragile NETs structures. Combined with the SYTOX™ Green single staining protocol, typical NETs structures were successfully induced in three independent replicate experiments. The images showed low background and high signal-to-noise ratio, demonstrating the excellent stability and reproducibility of the method.

[0042] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method of inducing HL-60 cells to produce neutrophil extracellular traps, comprising, comprising the steps of: providing HL-60 cells; inducing differentiation of the HL-60 cells into neutrophil-like cells using dimethyl sulfoxide; stimulating the neutrophil-like cells to induce formation of neutrophil extracellular traps using phorbol ester.

2. The method of claim 1, wherein, The final concentration of the dimethyl sulfoxide in the inducing differentiation step is 0.8% to 1.5% (v / v).

3. The method of claim 2, wherein, The final concentration of the dimethyl sulfoxide is 1.25% (v / v).

4. The method of claim 1, wherein, The final concentration of the phorbol ester in the stimulating step is 50 nM to 400 nM.

5. The method of claim 4, wherein, The final concentration of the phorbol ester is 100 nM.

6. The method of claim 1, wherein, The time of stimulating using phorbol ester is 2 to 4 hours.

7. The method of claim 6, wherein, The time of stimulating using phorbol ester is 3 to 4 hours.

8. The method of claim 1, wherein, The step of inducing formation of neutrophil extracellular traps is performed on a substrate coated with poly-lysine.

9. The method of claim 1, wherein, The formed neutrophil extracellular traps are fluorescently labeled and observed using SYTOX Green nucleic acid dye.

10. Use of the method of any one of claims 1 to 9 in the preparation of a reagent or model for drug screening, inflammation mechanism study or disease model construction.

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