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Mar 2020
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Analyzing the Quenchable Iron Pool in Murine Macrophages by Flow Cytometry
小鼠巨噬细胞可淬灭铁池的流式细胞分析   

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Abstract

Tissue-resident macrophages are pivotal for a tightly-regulated iron metabolism at a cellular and systemic level, since subtle iron alterations increase the susceptibility for microbial infections or drive multiple diseases. However, research on cellular iron homeostasis in macrophages remains challenging due to the limited amount of available methods using radioactive 59Fe isotopes or strong iron chelators, which might be inapplicable in certain experimental settings. This protocol describes the analysis of the quenchable iron pool (QIP) in macrophages by loading these cells with exogenous iron-complexes. Thereby, the cytoplasmic iron pool can be determined, since the iron uptake ability of macrophages inversely correlates with intracellular iron levels. Thus, this assay enables the accurate analysis of even minor alterations in cytoplasmic iron fluxes and is applicable in almost every laboratory environment. In addition, the protocol can also be adopted for other immune cell types in vitro and in vivo.

Keywords: Quenchable iron pool (淬火铁池), Labile iron pool (动态铁池), Cytoplasm (细胞质), Macrophage (巨噬细胞), Flow cytometry (流式细胞术), Calcein-AM (钙黄绿素-AM), 8-hydroxyquinoline (8-羟基喹啉), FeHQ (FeHQ)

Background

Owing to their central regulatory functions for systemic iron metabolism, macrophages in multiple tissues swiftly alter their intracellular iron levels upon encounter of diverse stimuli during microbial infections and diseases (Nairz et al., 2017). Of note, the majority of cellular iron ions is bound by the major iron storage protein Ferritin within the cytoplasm or is localized to compartments like mitochondria or lysosomes (Ma et al., 2015; Soares and Hamza, 2016). However, the labile iron pool represents an unbound fraction of cytoplasmic iron, which is metabolically accessible and pivotal for cellular iron homeostasis and metabolism (Cabantchik, 2014). Interestingly, subtle concentration changes within this macrophage iron pool can greatly impact the outcome of infections, especially with intracellular pathogens (Ganz and Nemeth, 2015).

However, these intracellular iron fluxes in the nanomolar to millimolar range are highly dynamic and are additionally regulated in a spatiotemporal manner, providing a significant challenge for accurate experimental quantification (Cabantchik, 2014; Ma et al., 2015). Methods for iron import/export studies, using radioactive 59Fe isotopes, are highly sensitive and specific, but are quite limited in their application for the broad interested scientific community owing to safety issues and governmental regulations. Further, quantification of chelated iron by spectroscopy is hampered by either physical cell disruption, low sensitivity or the large amounts of required biological materials.

To address these experimental challenges, Epsztejn et al. described a Calcein-AM-based method to analyze the labile iron pool (Epsztejn et al., 1997). When Calcein-AM is taken up by metabolically active cells, this acetoxy-methyl-ester is cleaved by intracellular esterases, releasing the iron-specific fluorescent dye Calcein exclusively into the cytoplasm (Ma et al., 2015). Thereby, Calcein fluorescence is quenched upon binding to cytoplasmic iron ions (Figure 1). Further, this method was refined for analysis by flow cytometry (Prus and Fibach, 2008). In general, these protocols rely on the usage of strong iron chelators to sequester cytoplasmic iron ions for subsequent Calcein fluorescence rescue. However, some iron chelators are commercially unavailable (e.g., salicylaldehyde isonicotinoyl hydrazone = SIH) or can have diminished chelating effects under specific experimental conditions.


Figure 1. Quenching of cellular Calcein fluorescence upon binding of iron ions. Calcein fluoresces in the unbound state but gets quenched during binding by accessible iron ions within the cytoplasm. Addition of exogenous iron upon treatment with FeCl2/8-hydroxyquinoline (FeHQ) complexes further quenches Calcein fluorescence.

Thus, in order to increase practical applicability, this Calcein-AM-based assay was modified by analyzing the quenchable iron pool (QIP) instead (Du et al., 2015; Siegert et al., 2015). Since Calcein shows reduced fluorescence in the iron-bound state, its fluorescence gets additionally quenched when exogenous iron is swiftly delivered into the cytoplasm by the addition of a FeCl2/8-hydroxyquinoline (FeHQ) complex (Figure 1) (Prachayasittikul et al., 2013; Ma et al., 2015; Chobot et al., 2018). Hence, the difference in cellular Calcein fluorescence before and after FeHQ addition represents the QIP (Figure 2A). Of note, the size of the unbound, cytoplasmic iron pool inversely correlates with the ability of a given cell to take up iron ions. Thus, iron-starved cells can substantially take up more exogenous Fe (upon FeHQ addition) than iron-saturated cells (Figure 2B). Therefore, cells with minor cytoplasmic iron pools exhibit higher QIPs when compared to iron-loaded cells with reduced QIPs (Figure 2A).

In conclusion, QIP analysis by flow cytometry represents a powerful tool to accurately determine alterations in cytoplasmic iron levels upon diverse cellular stimuli. Further, this protocol can be established easily in almost every laboratory environment and can also be applied for the investigation of iron homeostasis in other immune cell types in vitro and in vivo.


Figure 2. Theoretical concept of QIP analysis. A. Schematic representation of QIP calculation. Primary bone marrow-derived macrophages (BMDMs) were left untreated or treated with 50 μM FeSO4 for 17 h and the MFI (Calcein) was determined in naive and FeHQ-challenged BMDMs. The QIP at the respective condition is calculated as the MFI difference. B. The cytoplasmic levels of unbound iron inversely correlate with the cellular iron uptake ability. Thus, cells with low cytoplasmic iron levels can take up more exogenous iron (upon FeHQ treatment), than already iron-saturated cells. MFI, median fluorescence intensity.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml reaction tubes
  3. 50 ml conical centrifuge tubes (Starlab, catalog number: E1450-0200 )
  4. 15 ml conical centrifuge tubes (Starlab, catalog number: E1415-0200 )
  5. Tissue papers (Tork, catalog number: 290163 )
  6. 5 ml serological pipettes (Starlab, catalog number: E4860-0005 )
  7. 10 ml serological pipettes (Starlab, catalog number: E4860-0010 )
  8. 5 ml round-bottom FACS tubes, for example FalconTM Round-Bottom Polystyrene Tubes (Thermo Fisher Scientific, FalconTM, catalog number: 352054 )
  9. 6-well CytoOne® Plates, tissue culture-treated (Starlab, catalog number: CC7682-7506 )
  10. 24-well CytoOne® Plates, tissue culture-treated (Starlab, catalog number: CC7682-7524 )
  11. 10 cm round CytoOne® Dish, tissue culture-treated (Starlab, catalog number CC7682-3394 )
  12. T-150 CytoOne® Flask, tissue culture-treated, vented (Starlab, catalog number: CC7682-4815 )
  13. Thermo ScientificTM SterilinTM 100 mm Square Petri Dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 11349273 )
  14. Natural-rubber scraper (Deutsch & Neumann, catalog number: 2260001 )
  15. Rapid-FlowTM sterile disposable filter unit with cellulose nitrate-membrane: 0.2 μm pore size (Thermo ScientificTM, NalgeneTM, catalog number: 10261741 )
  16. Primary bone marrow-derived macrophages (BMDMs) from C57BL/6J mice: For BMDM cultivation in general, please refer to Bourgeois et al., 2009
  17. L929 cell line (ATCC, catalog number: CCL-1 )
  18. Purified anti-mouse CD16/32 antibody (BioLegend, catalog number: 101302 )
  19. FITC anti-mouse/human CD11b antibody (BioLegend, catalog number: 101205 )
  20. PE anti-mouse F4/80 antibody (BioLegend, catalog number: 123110 )
  21. Ultra-pure, high quality water, suitable for trace metal analysis (Sigma-Aldrich, catalog number: 14211 )
  22. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11584486 )
  23. Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: F7524 )
  24. Penicillin/Streptomycin (Sigma-Aldrich, catalog number: P4333 )
  25. Phosphate-buffered saline (PBS), Ca2+ and Mg2+ free, liquid (Sigma-Aldrich, catalog number: D8537 )
  26. FeCl2∙4H2O (Sigma-Aldrich, catalog number: 220299 )
  27. 8-hydroxyquinoline (Sigma-Aldrich, catalog number: H6878 )
  28. Calcein-AM (BioLegend, catalog number: 425201 )
  29. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2438 )
  30. Trypsin solution 10x (Sigma-Aldrich, catalog number: 59427C )
  31. EDTA disodium salt dihydrate (AppliChem, catalog number: 131669.1211 )
  32. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  33. Complete DMEM (see Recipes)
  34. L929-conditioned medium (see Recipes)
  35. BMDM medium (see Recipes)
  36. Stock solution preparation of Calcein-AM, FeCl2 and 8-hydroxyquinoline (see Recipes)
  37. Calcein-AM staining solution (see Recipes)
  38. FeHQ solution (see Recipes)
  39. Trypsin solution (see Recipes)
  40. FACS buffer (see Recipes)

Equipment

  1. Pipettes, for example Pipetman model (Gilson)
  2. Motorized single-channel pipette (Gilson, Pipetman® Concept 100-1,200 μl)
  3. Combined orbital linear shaking water bath (Grant Instruments, model: OLS200 )
  4. Laminar flow hood suitable for cell culture, for example Microbiological Safety Cabinets (Szabo-Scandic, SafeFAST Premium 212)
  5. CO2-incubator (Binder, model: CB 170 )
  6. Automated cell counter, for example CASY cell counter & analyzer (Roche, model: TTC-2KA-2037 )
  7. Refrigerated centrifuge (Hettich, model: Rotina 38R )
  8. Refrigerated bench top centrifuge (Peqlab, PerfectSpin 24R Refrigerated Microcentrifuge)
  9. Flow cytometer equipped with appropriate lasers and filters for the detection of Calcein fluorescence, for example BD LSRFortessaTM (BD Biosciences)

Software

  1. BD FACSDiva Software (BD Biosciences, Version: 8.0.1)
  2. FlowJo Software (FlowJo, Version: 7.6.5)

Procedure

  1. BMDM harvest and preparation
    1. For differentiation of primary bone marrow-derived macrophages (BMDMs), cultivate murine bone marrow cells (isolated from one tibia and femur) in BMDM medium and 10 cm square tissue culture dishes for 10 days at 37 °C, 5% CO2.
      Note: For BMDM cultivation in general, please refer to Bourgeois et al. (2009).
    2. Harvest BMDMs from 10 cm square dishes by gently scraping with a natural-rubber scraper 24 h before the experiment on Day 10 of cultivation and centrifuge at 300 x g, 20 °C for 6.5 min.
    3. Aspirate medium and resuspend the BMDM pellet in 1 ml BMDM medium.
    4. Accurately determine the BMDM cell concentration by using an automated cell counter.
    5. Transfer 2.5 x 105 BMDMs into a 24-well plate, supplied with a total volume of 300 μl BMDM medium.
      Note: Per every experimental condition, at least 3 technical replicates each for untreated and FeHQ-treated BMDMs have to be prepared (in total: minimum 6 x 24-wells/condition). Further, include one replicate of untreated BMDMs as unstained control for flow cytometry analysis.
    6. Incubate BMDMs overnight at 37 °C, 5% CO2.

  2. BMDM stimulation
    1. Stimulate BMDMs with the respective reagent (e.g., LPS, cytokines, bacterial or fungal pathogens) under the desired condition.
    2. Incubate BMDMs at 37 °C, 5% CO2.

  3. BMDM staining and preparation for flow cytometry analysis
    1. Thirty minutes before the end of BMDM stimulation, warm PBS to 37 °C in a water bath, freshly prepare FeHQ solution in sufficient amounts and adjust all required materials for subsequent BMDM staining procedure.
    2. Immediately before the end of the BMDM experiment, prepare the Calcein-AM staining solution and store it in the dark.
      Note: Calcein-AM is highly susceptible for hydrolysis in aqueous buffer solutions. Therefore, to ensure maximal BMDM staining efficiency, work as quick and as accurate as possible from this step on.
    3. Remove the BMDM medium by quickly tilting the 24-well plate onto a stack of tissue papers.
    4. Wash BMDMs 3x with 300 μl pre-warmed PBS and remove the wash solution by quickly tilting the 24-well plate onto the used stack of tissue papers.
      Note: Completely remove FCS-containing cell culture medium, since FCS residuals alter Calcein-AM staining of BMDMs. Always dispose the used stack of tissue papers into the corresponding safety waste.
    5. Add 300 μl Calcein-AM staining solution/well with a motorized single-channel pipet and incubate BMDMs at 37 °C, 5% CO2 for 15 min.
    6. Remove the Calcein-AM staining solution by quickly tilting the 24-well plate onto a fresh stack of tissue papers.
    7. Wash BMDMs 3x with 300 μl pre-warmed PBS and remove the wash solution by quickly tilting the 24-well plate onto the used stack of tissue papers.
    8. Add 300 μl PBS to BMDMs of the untreated condition and 300 μl FeHQ solution to BMDMs of the FeHQ-treated condition with a motorized single-channel pipet and incubate BMDMs at 37 °C, 5% CO2 for 30 min.
      Note: BMDM incubation in FeHQ solution longer than 30 min will detrimentally affect BMDM membrane integrity. Therefore, subsequent BMDM harvest and flow cytometry analysis should also be performed as soon as possible.
    9. Remove the solutions by quickly tilting the 24-well plate onto a fresh stack of tissue papers.
    10. Wash BMDMs 3x with 300 μl pre-warmed PBS and remove the wash solution by quickly tilting the 24-well plate onto the used stack of tissue papers
    11. Add 250 μl trypsin solution per 24-well with a motorized single-channel pipet and incubate BMDMs at 37 °C, 5% CO2 for 5 min.
    12. Place 24-well plate on ice and add 750 μl ice-cold complete DMEM to the wells in order to prevent further trypsinization.
    13. Detach BMDMs by pipetting up and down with a 1,000 μl single-channel pipet, transfer the cell suspension of every technical replicate to a separate 1.5 ml reaction tube and store on ice in the dark.
    14. After harvesting all BMDMs, centrifuge cell suspensions at 400 x g, 4 °C for 5 min.
    15. Aspirate medium, resuspend BMDM pellet in 300 μl FACS buffer and transfer to 5 ml round-bottom FACS tubes.
    16. Process all BMDM samples and store FACS tube on ice in the dark.
    17. Analyze BMDM samples on a flow cytometer equipped with appropriate lasers and filters for the detection of Calcein fluorescence with excitation maximum ~490 nm and emission maximum ~510 nm (Sabnis, 2010) as soon as possible.

Data analysis

  1. Using appropriate flow cytometry analysis software, display the samples on a two-dimensional dot plot of FSC-A vs. SSC-A and gate on the BMDM population (Figure 3A).
  2. For doublet discrimination, gate on single cells on an FSC-A vs. FSC-H dot plot (Figure 3A).
  3. By measuring the unstained control sample, define unstained and Calcein+ cells on a Calcein histogram (Figure 3A).
  4. Analyze all BMDM samples by recording the Calcein fluorescence.
  5. Determine the MFI (Calcein) (median fluorescence intensity) of Calcein+ BMDMs.
  6. Calculate the QIP as difference in MFI (Calcein) between untreated and FeHQ-treated BMDMs per every experimental condition:

    QIP = MFI(untreated cells) - MFI(FeHQ-treated cells)

  7. Visualize QIPs of BMDMs at different conditions in a column graph and perform statistical analysis (Figure 3B).


    Figure 3. QIP analysis in BMDMs upon infectious stimuli. A. Gating strategy for QIP analysis. After doublet discrimination on an FSC-A vs. FSC-H dot plot, the MFI (Calcein) of Calcein+ cells is recorded. B. BMDMs were infected with a MOI = 5 (pathogen:host cell ratio = 5:1) of heat-killed (70 °C for 10 min) Candida albicans (Ca) or Candida glabrata (Cg) for 12 h and subsequently analyzed for QIP alterations. Thereby, BMDMs upregulated their cytoplasmic iron levels during challenge with Candida spp., owing to the reduced QIPs upon infectious stimuli. MFI, median fluorescence intensity (*** P-value < 0.001).

Notes

  1. The FeHQ solution should be adjusted approximately 30 min before BMDM incubation with Calcein-AM staining solution and should be mixed several times in between to ensure adequate complex formation of Fe2+ ions and 8-hydroxyquinoline.
  2. As mentioned before, the Calcein-AM staining solution should be freshly prepared directly before the end of BMDM stimulation and immediately be used for subsequent BMDM staining owing to the high hydrolysis susceptibility of Calcein-AM in aqueous solutions. For example, 10 min in aqueous buffers decreases the BMDM staining efficiency of Calcein-AM for approximately 50%.
  3. Harvested BMDM samples should be analyzed by flow cytometry as soon as possible, since FeHQ loading of BMDMs shows adverse effects on cell membrane integrity upon prolonged storage of already processed samples.
  4. This assay is highly accurate and the observed biological effects in BMDMs can be well repeated. However, the general amplitude of QIPs can vary between independent biological experiments, since the QIP is a function of Calcein fluorescence and, therefore, depends on the Calcein-AM staining efficiency of BMDMs.
  5. For iron uptake studies into the cytoplasm, BMDMs should only be treated with solutions, for which the iron-containing chemicals have been diluted in ultra-pure, high quality water with minimal trace metal concentrations (e.g., grades suitable for trace element analysis). Otherwise, Fe2+/Fe3+ will form water-insoluble complexes in phosphate-buffered solutions and, therefore, will distort iron uptake by BMDMs.
  6. This protocol can also be applied for other immune cell types in vitro and in vivo. Thereby, aliquots of 1 x 106 from the desired cell type (e.g., non-adherent cells or in vivo cell populations) should be transferred into 1.5 ml reaction tubes and incubated with 200 μl Calcein-AM staining solution respectively FeHQ solution at 37 °C in a bench top thermomixer without agitation in the dark. The number of PBS washing steps in between can be reduced. However, in a pilot experiment, the required final Calcein-AM concentration, FeHQ composition and the incubation time for FeHQ loading should be optimized in detail for every cell type of interest via titration.

Recipes

  1. Complete DMEM
    1. Add 10% heat-inactivated fetal calf serum (hiFCS) and 1x Penicillin/Streptomycin to DMEM
    2. Store at 4 °C for up to one month
    Note: For heat inactivation, incubate thawed FCS in a water bath at 56 °C for 30 min and invert FCS bottle every 10 min. hiFCS can be stored in aliquots at -20 °C for up to 6 months.
  2. L929-conditioned medium
    Preparation of L929-conditioned medium
    Note: The following protocol for preparation of L929-conditioned medium was described previously (Bourgeois et al., 2009).
    1. Cultivate M-CSF-producing L929 cells in 12 ml complete DMEM and 10 cm round tissue culture dishes until confluency at 37 °C, 5% CO2
    2. Harvest L929 cells from one 10 cm round tissue culture dish by gently scraping with a natural-rubber scraper and transfer the cell suspension into one T-150 cell culture flask with 80 ml complete DMEM
    3. Incubate L929 cells for 3 days until confluency at 37 °C, 5% CO2
    4. Aspirate the medium and add 100 ml DMEM per every T-150 cell culture flask
      Note: At this step, it is crucial to use DMEM (without hiFCS) instead of complete DMEM (supplemented with hiFCS).
    5. Incubate L929 cells for 10 days at 37 °C, 5% CO2
    6. Harvest the supernatants and filter them through a sterile disposable filter unit
    7. Store 50 ml aliquots at -20 °C for up to 12 months

    Testing of L929-conditioned medium
    1. Cultivate murine bone marrow cells in 3 ml complete DMEM with increasing concentrations of L929-conditioned medium (0-20%) in 6-well plates and incubate for 8 days at 37 °C, 5% CO2
      Note: L929-conditioned medium from previous, already tested batches (or commercially available recombinant M-CSF) can be used as positive control for macrophage differentiation.
    2. Harvest adherent BMDMs by trypsinization and determine cellular yield of BMDMs by using an automated cell counter
    3. Incubate BMDMs with antibodies against CD16/32 and macrophage-specific cell surface markers (e.g., CD11b, F4/80) to verify macrophage differentiation by FACS analysis
      Note: BMDM differentiation via cultivation of bone marrow cells in L929-conditioned medium yields ~98% CD11b+ F4/80+ macrophages.
    4. Deduce from cellular yield and FACS analysis the optimal concentration of L929-conditioned medium for BMDM differentiation
  3. BMDM medium
    1. Add 15% L929-conditioned medium to complete DMEM
    2. Store at 4 °C for up to one month
  4. Stock solution preparation
    1. For Calcein-AM stocks, dissolve 50 μg solid Calcein-AM in 50 μl anhydrous DMSO to prepare a 1 mM stock. Freeze aliquots in amber reaction tubes at -20 °C protected from light for up to 4 months. Avoid multiple freezing/thawing cycles
    2. For FeCl2 stocks, dissolve FeCl2 in ultra-pure, high quality water and freeze 1 mM stocks at -20 °C
    3. For 8-hydroxyquinoline stocks, dissolve 8-hydroxyquinoline in anhydrous DMSO, prepare 200 μM stocks by dilution with PBS and store aliquots at -20 °C
  5. Calcein-AM staining solution
    1. Dilute 1 μl of 1 mM Calcein-AM stock (f.c. 1 μM) in 1 ml PBS, vortex and store the solution in the dark until use
    2. Immediately incubate BMDMs in freshly prepared Calcein-AM staining solution to ensure maximum BMDM staining efficiency
  6. FeHQ solution
    1. Add 5 μl of 1 mM FeCl2 (f.c. 5 μM) to 945 μl PBS and vortex
    2. Subsequently, add 50 μl of 200 μM 8-hydroxyquinoline (f.c. 10 μM) and vortex
    3. Until BMDM treatment, mix FeHQ solution every 10 min to ensure adequate complex formation of Fe2+ ions and 8-hydroxyquinoline
  7. Trypsin solution
    1. Add 500 μl of 10x trypsin to 4.5 ml PBS and 100 μl of 500 mM EDTA (pH = 8.0)
      Note: For trypsinization in general, it is crucial to use Ca2+ and Mg2+ free PBS.
    2. Incubate trypsin solution in a water bath at 37 °C until use
  8. FACS buffer
    1. Dissolve 0.5 g BSA (f.c. 1%) in 50 ml PBS
    2. Filter-sterilise and store FACS buffer at 4 °C for up to 1 month

Acknowledgments

This protocol was supported by the Austrian Science Fund project InnateFun (FWF-I3319-B22) and in part by the project FUNGITECT (HEALTH-F3-2013-602125) from the EC-FP7 program to KK. We would like to acknowledge the studies from Du et al. (2015) and Siegert et al. (2015) for establishing this elegant method.

Competing interests

The authors declared that no competing interests exist.

References

  1. Bourgeois, C., Majer, O., Frohner, I., and Kuchler, K. (2009). In vitro systems for studying the interaction of fungal pathogens with primary cells from the mammalian innate immune system. Methods Mol Biol 470: 125-139.
  2. Cabantchik, Z. I. (2014). Labile iron in cells and body fluids: physiology, pathology, and pharmacology. Front Pharmacol 5: 45.
  3. Chobot, V., Hadacek, F., Bachmann, G., Weckwerth, W. and Kubicova, L. (2018). Antioxidant properties and the formation of iron coordination complexes of 8-hydroxyquinoline. Int J Mol Sci 19(12). 
  4. Du, J., Wagner, B. A., Buettner, G. R. and Cullen, J. J. (2015). Role of labile iron in the toxicity of pharmacological ascorbate. Free Radic Biol Med 84: 289-295.
  5. Epsztejn, S., Kakhlon, O., Glickstein, H., Breuer, W. and Cabantchik, I. (1997). Fluorescence analysis of the labile iron pool of mammalian cells. Anal Biochem 248(1): 31-40.
  6. Ganz, T. and Nemeth, E. (2015). Iron homeostasis in host defence and inflammation. Nat Rev Immunol 15(8): 500-510. 
  7. Ma, Y., Abbate, V. and Hider, R. C. (2015). Iron-sensitive fluorescent probes: monitoring intracellular iron pools. Metallomics 7(2): 212-222. 
  8. Nairz, M., Theurl, I., Swirski, F.K., and Weiss, G. (2017). “Pumping iron”—how macrophages handle iron at the systemic, microenvironmental, and cellular levels. Pflugers Arch 469: 397-418.
  9. Prachayasittikul, V., Prachayasittikul, S., Ruchirawat, S., and Prachayasittikul, V. (2013). 8-Hydroxyquinolines: a review of their metal chelating properties and medicinal applications. Drug Des Devel Ther 7: 1157-1178.
  10. Prus, E., and Fibach, E. (2008). Flow cytometry measurement of the labile iron pool in human hematopoietic cells. Cytometry A 73(1): 22-27.
  11. Sabnis, R. (2010). Handbook of biological dyes and stains: synthesis and industrial applications Wiley Inc. USA. ISBN: 978-0-470-40753-0.
  12. Siegert, I., Schodel, J., Nairz, M., Schatz, V., Dettmer, K., Dick, C., Kalucka, J., Franke, K., Ehrenschwender, M., Schley, G., Beneke, A., Sutter, J., Moll, M., Hellerbrand, C., Wielockx, B., Katschinski, D. M., Lang, R., Galy, B., Hentze, M. W., Koivunen, P., Oefner, P. J., Bogdan, C., Weiss, G., Willam, C. and Jantsch, J. (2015). Ferritin-mediated iron sequestration stabilizes hypoxia-inducible factor-1α upon LPS activation in the presence of ample oxygen. Cell Rep 13(10): 2048-2055. 
  13. Soares, M. P. and Hamza, I. (2016). Macrophages and iron metabolism. Immunity 44(3): 492-504.

简介

[摘要 ] 驻留在组织中的巨噬细胞对于在细胞和全身水平上严格调节铁代谢至关重要,因为细微的铁改变会增加对微生物感染的敏感性或引发多种疾病。然而,由于使用放射性59 Fe同位素或强铁螯合剂的可用方法数量有限,因此巨噬细胞中细胞铁稳态的研究仍然具有挑战性,这在某些实验环境中可能不适用。该协议 描述了通过向这些细胞加载外源铁配合物来分析巨噬细胞中的可淬灭铁池(QIP)。因此,由于巨噬细胞的铁摄取能力与细胞内铁水平成反比,因此可以确定细胞质的铁库。因此,该测定法能够准确分析细胞质铁通量的微小变化,并且几乎适用于所有实验室环境。另外,该方案还可以用于体外和体内的其他免疫细胞类型。

[背景 ] 由于用于全身铁代谢它们的中心调节功能,在多种组织中的巨噬细胞中迅速地改变在不同的刺激的遭遇它们的细胞内的铁水平的微生物感染和疾病(Nairz 等人,2017) 。值得注意的是,大多数细胞中的铁离子被细胞质中的主要铁存储蛋白铁蛋白结合或定位于线粒体或溶酶体等区室(Ma 等,2015 ; Soares和Hamza,2016 )。然而,不稳定的铁池代表了细胞质铁的未结合部分,其可以通过代谢途径获得,并且对于细胞铁稳态和代谢至关重要(Cabantchik,2014)。有趣的是,该巨噬细胞铁库中的细微浓度变化会极大地影响感染的结果,尤其是细胞内病原体的感染(Ganz和Nemeth,2015)。

然而,这些纳摩尔至毫摩尔范围内的细胞内铁通量是高度动态的,并以时空方式进行调节,为准确的实验定量化提出了重大挑战(Cabantchik,2014; Ma et al。,2015)。使用放射性59 Fe同位素进行铁进出口研究的方法高度敏感且特异,但由于安全问题和政府法规,其在广泛感兴趣的科学界中的应用受到很大限制。此外,通过物理细胞破坏,低灵敏度或大量所需的生物材料阻碍了通过光谱法对螯合铁的定量。

为了解决这些实验挑战,Epsztejn 等。述的钙黄绿素的基于-AM-方法ANALY Ž Ë 不稳定铁池(Epsztejn 等人,1997) 。当钙黄绿素-AM是采取了由代谢活性细胞中,该乙酰氧基甲基酯是由细胞内裂解酯酶,释放铁特异性荧光染料钙黄绿素只进入细胞质(马等人,2015) 。因此,钙黄绿素荧光在与细胞质铁离子结合后被淬灭(图1)。此外,该方法被改进用于流式细胞术分析(Prus和Fibach,2008)。通常,这些协议依赖于使用强铁螯合剂来隔离细胞质铁离子,以用于随后的钙黄绿素荧光拯救。但是,某些铁螯合剂在商业上不可用(例如,水杨醛异烟酰yl = SIH)或在特定实验条件下螯合作用减弱。





图1. 铁离子结合后细胞钙黄绿素荧光的猝灭。钙黄绿素以未结合状态发荧光,但在结合过程中被细胞质内可及的铁离子淬灭。在用的FeCl治疗外源性铁的另外2 /8-羟基喹啉(FeHQ )络合物进一步猝灭钙黄绿素荧光。



因此,为了提高实用性,这钙黄绿素基于-AM-测定通过改性ANALY Ž 荷兰国际集团的淬火铁池(QIP)代替(都等人,2015;西格特。等人,2015) 。由于钙黄绿素在铁结合状态下显示出减少的荧光,因此当通过添加FeCl 2 / 8-羟基喹啉(FeHQ )络合物将外源铁迅速递送到细胞质中时,其荧光会进一步淬灭(图1)(Prachayasittikul 等。 ,2013; Ma 等,2015; Chobot 等,2018)。因此,在蜂窝的差异钙黄绿素之前和之后荧光FeHQ 除了表示QIP(图2A)。值得注意的是,未结合的细胞质铁池的大小与给定细胞吸收铁离子的能力成反比。因此,铁缺乏的细胞比铁饱和的细胞可以吸收更多的外源铁(FeHQ 添加后)(图2B)。因此,与具有降低的QIP的载铁细胞相比,具有较小胞质铁池的细胞表现出更高的QIP(图2A)。

总之,通过流式细胞仪进行的QIP分析是一种功能强大的工具,可以根据各种细胞刺激物准确确定细胞质中铁水平的变化。此外,该方案可以在几乎每个实验室环境中轻松建立,也可以用于体外和体内其他免疫细胞类型中铁稳态的研究。





图2. QIP分析的理论概念。一。QIP计算的示意图。原代骨髓源巨噬细胞(的BMDM)不处理或用50处理μM 的FeSO 4 17小时和MFI (钙黄绿素)的幼稚测定并FeHQ -challenged的BMDM。计算相应条件下的QIP作为MFI差。B. 未结合的铁的细胞质水平与细胞吸收铁的能力成反比。因此,与已经饱和铁的细胞相比,具有低胞质铁水平的细胞可以吸收更多的外源铁(FeHQ 处理)。MFI,中值荧光强度。

关键字:淬火铁池, 动态铁池, 细胞质, 巨噬细胞, 流式细胞术, 钙黄绿素-AM, 8-羟基喹啉, FeHQ

材料和试剂


 


移液器技巧
1.5 ml反应管
50 ml锥形离心管(Starlab ,目录号:E1450-0200)
15 ml锥形离心管(Starlab ,目录号:E1415-0200)
薄纸(Tork ,目录号290163)
5 ml血清移液器(Starlab ,目录号:E4860-0005)
10 ml血清移液器(Starlab ,目录号:E4860-0010)
5 ml圆底FACS管,例如Falcon TM 圆底聚苯乙烯管(Thermo Fisher Scientific,Falcon TM ,目录号:352054)
6- 瓦特ELL CytoOne ® 板,组织培养物处理的(STARLAB ,目录号码:CC7682-7506)
24- 瓦特ELL CytoOne ® 板,组织培养物处理的(STARLAB ,目录号码:CC7682-7524)
10厘米轮CytoOne ® 盘子,组织培养物处理的(STARLAB ,目录号CC7682-3394)
T-150 CytoOne ® 烧瓶中,组织培养物处理过的,通风(STARLAB ,目录号码:CC7682-4815)
热科学TM Sterilin TM 100 毫米方培养皿(赛默飞世尔科技,热科学TM ,目录号:11349273)
天然橡胶刮板(Deutsch&Neumann,目录号:2260001)
速效流TM 无菌一次性过滤器单元与硝酸纤维素膜:0.2 微米孔径(热科学TM ,的Nalgene TM ,目录号:10261741)
来自C57BL / 6J小鼠的原代骨髓巨噬细胞(BMDM):有关BMDM培养的一般信息,请参阅Bourgeois 等,20 09
L929细胞系(ATCC,目录号:CCL-1)
纯化的抗小鼠CD16 / 32抗体(BioLegend公司,目录号:101302)
FITC抗小鼠/人类CD11b抗体(BioLegend ,目录号:101205)
PE抗小鼠F4 / 80抗体(BioLegend公司,目录号:123110)
超纯优质水,适用于痕量金属分析(Sigma-Aldrich,目录号:14211)
DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11584486)
胎儿小牛血清(FCS)(Sigma-Aldrich公司,目录号:F7524)
青霉素/链霉素(西格玛奥德里奇,目录号:P4333)
磷酸盐缓冲盐水(PBS),不含Ca 2+ 和Mg 2+ ,液体(Sigma-Aldrich,目录号:D8537)
FeCl 2 ∙4H 2 O(Sigma-Aldrich ,目录号:220299)
8-羟基喹啉(Sigma-Aldrich ,目录号:H6878)
钙黄绿素-AM(BioLegend公司,目录号:425201)
二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2438 )
胰蛋白酶溶液10x(Sigma-Aldrich,目录号:59427C)
EDTA二钠二水合物盐(AppliChem,目录号:131669.1211)
牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
完整的DMEM(请参阅食谱)
L929条件培养基(请参阅食谱)
BMDM介质(请参阅食谱)
的储备溶液制备钙黄绿素-AM,的FeCl 2 和8-羟基喹啉(见配方)
钙黄绿素-AM染色溶液(见食谱)
FeHQ 解决方案(请参阅食谱)
胰蛋白酶溶液(请参阅食谱)
FACS缓冲区(请参阅配方)
 


设备


 


移液器,例如Pipetman 模型(Gilson)
电动单通道移液器(吉尔森的Pipetman ® 概念100-1 ,200 微升)
组合式轨道线性摇动水浴箱(格兰特仪器公司,型号:OLS200)
适用于细胞培养的层流罩,例如微生物安全柜(Szabo- Scandic ,SafeFAST Premium 212)
CO 2 培养箱(活页夹,型号:CB 170)
自动细胞计数器,例如CASY细胞计数器和分析仪(罗氏,型号:TTC-2KA-2037)
冷冻离心机(海蒂诗,型号:Rotina 38R)
冷藏台式离心机(Peqlab ,PerfectSpin 24R冷藏微量离心机)
流式细胞仪配备有适当的激光器和滤波器,用于检测的钙黄绿素的荧光,例如BD LSRFortessa TM (BD Biosciences)中
 


软件


 


BD FACSDiva 软件(BD Biosciences,版本:8.0.1)
FlowJo 软件(FlowJo ,版本:7.6.5)
 


程序


 


BMDM收获和准备
为了区分原代骨髓来源的巨噬细胞(BMDM),在BMDM培养基和10 cm正方形组织培养皿中于37°C,5 %CO 2下培养鼠骨髓细胞(从一个胫骨和股骨中分离)10天。
注意:有关BMDM的一般培养,请参阅Bourgeois等。(2009)。


从10厘米见方的培养皿收获的BMDM通过用刮轻轻天然橡胶刮板24 在实验之前ħ d 着y培养10 和离心机在300 X 克,20℃6.5分钟。
吸出培养基并将BMDM沉淀重悬于1 ml BMDM培养基中。
通过使用自动细胞计数器准确确定BMDM细胞浓度。
转移2.5×10 5 的BMDM到24孔板中,用300总体积供给微升BMDM平台。
注意:对于每种实验条件,必须准备至少3份未经处理的和FeHQ 处理的BMDM的技术重复样品(总计:最少6 x 24孔/条件)。此外,包括一份未经处理的BMDM复制品作为流式细胞仪分析的未染色对照。


将BMDM 在37°C,5 %CO 2 下孵育过夜。
 


BMDM刺激
刺激的BMDM与相应的试剂(例如,所希望的条件下的LPS,细胞因子,细菌或真菌病原体)。
在37°C,5 %CO 2 下孵育BMDM 。
 


BMDM染色和流式细胞仪分析准备
在BMDM刺激结束前30分钟,在水浴中将PBS加热至37°C,新鲜制备足够量的FeHQ 溶液,并调整所有必需的材料以用于随后的BMDM染色程序。
紧随BMDM实验结束前,准备黄绿素-AM染色解决方案,并将其存储在黑暗中。
注意:钙黄绿素-AM为在水性缓冲溶液水解高度敏感。因此,为了确保最大的BMDM染色效率,请在此步骤之前尽快并尽可能准确地工作。


通过将24孔板快速倾斜到一叠薄纸上,除去BMDM介质。
洗3次的BMDM用300 μL 预加热的PBS并通过快速倾斜24孔板上的薄棉纸所使用的堆栈除去洗涤溶液中。
注:完全去除含FCS细胞培养基,因为FCS残差改变黄绿素-AM染色的BMDM的。始终将用过的纸巾叠丢入相应的安全废物中。


添加300 微升钙黄绿素-AM染色溶液/孔与机动单通道移液器和孵化的BMDM在37℃,5 %CO 2 15分钟。
除去钙黄绿素通过快速倾斜24孔板上的薄棉纸新鲜堆-AM染色溶液。
洗3次的BMDM用300 μL 预加热的PBS并通过快速倾斜24孔板上的薄棉纸所使用的堆栈除去洗涤溶液中。
添加300 微升PBS中,以未处理的条件和300的的BMDM 微升FeHQ 解决的的BMDM FeHQ 在治疗过的条件与机动单通道移液器孵育的BMDM 37℃,5 %CO 2 30分钟。
注意:BMDM在FeHQ 溶液中孵育超过30分钟将对BMDM膜的完整性产生不利影响。因此,随后的BMDM收获和流式细胞仪分析也应尽快进行。


通过将24孔板快速倾斜到新鲜的薄纸叠上,除去溶液。
洗3次的BMDM用300 μL 预温PBS和通过快速倾斜24孔板上的薄棉纸所使用的堆栈除去洗涤溶液中的
使用电动单通道移液器每24孔添加250μl 胰蛋白酶溶液,并将BMDM在37°C,5 %CO 2 下孵育5分钟。
地点24孔板在冰上,并添加750 微升冰冷却的完全DMEM 中以防止进一步的胰蛋白酶消化到孔中。
分离的BMDM通过上下吹打以1 ,000 微升单通道移液管,在黑暗中的每个技术复制的细胞悬浮液转移到一个分开的1.5ml反应管中并储存在冰上。
收获所有BMDM后,将悬浮液在400 x g ,4°C下离心5分钟。
吸出培养基,重悬沉淀BMDM在300 微升FACS缓冲液,并转移至5ml圆底FACS管。
处理所有BMDM样品,并将FACS管放在黑暗中的冰上。
ANALY ž Ë 在配备有合适的激光器和滤波器用于检测的流式细胞仪BMDM样品钙黄绿素的荧光与激发最大值〜490nm和发射最大值〜510nm的(Sabnis,2010)尽快。
 


数据分析


 


使用适当的流式细胞仪分析软件,在FSC-A与SSC-A的二维点图上显示样品,并在BMDM群体上显示门(图3A)。
为了区分双峰,请在FSC-A与FSC-H点图上对单个单元进行门控(图3A)。
通过测量未染色的对照样品,在钙黄绿素直方图上定义未染色和钙黄绿素+ 细胞(图3A)。
ANALY ž Ë 通过记录所有BMDM样品钙黄绿荧光。
确定MFI (钙黄绿素的)(中位数荧光强度)的钙黄绿素+ 的BMDM。
计算QIP如MFI差(钙黄绿素未处理的和之间)FeHQ -处理每每个实验条件下的BMDM:
 


QIP = MFI (未处理的细胞) - MFI (FeHQ 处理的细胞)


 


在柱状图中可视化BMDM在不同条件下的QIP,并进行统计分析(图3B)。
 






图3.感染后在BMDM中的QIP分析。一。QIP 分析的门控策略。在n FSC-A对FSC-H点图上进行双重鉴别后,记录钙黄绿素+ 细胞的MFI (钙黄绿素)。B. 的BMDM感染了一个MOI = 5(病原体:宿主细胞比= 5:1)热灭活的(70℃,10分钟)白色念珠菌(钙)或光滑念珠菌(CG)12小时并随后被分析用于QIP更改。因此,由于在感染刺激下降低的QIP,BMDMs在用假丝酵母刺激的过程中上调了其胞质铁水平。MFI,中值荧光强度(*** P 值<0.001)。


 


笔记


 


该FeHQ 溶液应该BMDM孵育之前调整大约30分钟钙黄绿素-AM染色溶液,并应在之间,以确保铁充足的复合物形成混合几次2+ 离子和8-羟基喹啉。
如之前提到的那样,钙黄绿素-AM染色溶液应新鲜直接制备BMDM刺激结束前,并立即用于随后的染色BMDM由于高水解敏感性钙黄绿素在水溶液中-AM。例如,10 在水性缓冲液分钟下降š 的BMDM染色效率的钙黄绿素-AM对大约50 %。
收获BMDM样品应该是ANALY Ž ED 通过流式细胞术尽快,由于FeHQ 的的BMDM示出在延长的细胞膜完整性的不利影响载荷已加工的样品的存储。
该测定法非常准确,并且可以很好地重复观察到的BMDM中的生物学效应。然而,速效项目的一般振幅可以独立生物实验之间变化,由于QIP是的函数钙黄绿素的荧光,因此,依赖于钙黄绿素的BMDM的-AM染色效率。
为了研究铁对细胞质的吸收,仅应使用溶液处理BMDM,其中含铁化学物质已在超纯,高质量水中以最低的痕量金属浓度(例如,适用于痕量元素分析的等级)稀释。否则,Fe 2+ / Fe 3+ 将在磷酸盐缓冲溶液中形成水不溶性复合物,因此会扭曲BMDM吸收铁。
该方案还可以应用于体外和体内的其他免疫细胞类型。从而,1等分试样X 10 6 从所需细胞类型(例如,非贴壁细胞或体内的细胞群)应被转移到1.5ml反应管中,用200孵育微升钙黄绿素-AM染色溶液分别FeHQ 在37°溶液C在台式热混合器中,无需在黑暗中搅拌。可以减少之间的PBS洗涤步骤。然而,在预实验中,所需的最终钙黄绿素-AM浓度,FeHQ 组成和温育时间为FeHQ 装载应进行详细用于通过滴定每个感兴趣的细胞类型优化。
 


菜谱


 


完整的DMEM
向DMEM中添加10 %热灭活的胎牛血清(hiFCS )和1x 青霉素/链霉素
在4°C下储存最多一个月
注意:对于热灭活,将融化的FCS在56°C的水浴中孵育30分钟,每10分钟翻转FCS瓶一次。hiFCS 可以等分保存在-20°C下长达6个月。


L929条件培养基
 


L929条件培养基的制备


注意:以下描述了用于制备L929条件培养基的方案(Bourgeois等,2009)。


在12 ml完整DMEM和10 cm圆形组织培养皿中培养产生M-CSF的L929细胞,直到在37°C,5 %CO 2 汇合
通过用天然橡胶刮板轻轻刮擦,从一个10厘米圆形组织培养皿中收获L929细胞,并将细胞悬浮液转移到一个装有80 ml完整DMEM的T-150细胞培养瓶中
将L929细胞孵育3天,直到在37°C,5 %CO 2 汇合时
吸出培养基,每个T-150细胞培养瓶中加入100 ml DMEM
注意:在此步骤中,至关重要的是使用DMEM(不带hiFCS )而不是完整的DMEM(由hiFCS 补充)。


将L92 9细胞在37°C,5 %CO 2 下孵育10天
收集上清液,并通过无菌一次性过滤器过滤
将50 ml等分试样在-20°C下存储最多12个月
 


L929条件培养基的测试


在6孔板中的3 ml完整DMEM中用浓度递增的L929条件培养基(0-20 %)培养鼠骨髓细胞,并在37°C,5 %CO 2 下孵育8天
注意:来自先前已测试批次的L929条件培养基(或市售的重组M-CSF)可用作巨噬细胞分化的阳性对照。


通过胰蛋白酶消化收集粘附的BMDM,并通过使用自动细胞计数器确定BMDM的细胞产量
孵育的BMDM用抗CD16 / 32抗体和巨噬细胞特异性细胞表面标记物(例如,细胞CD11b,F4 / 80),以验证巨噬细胞d 通过FACS分析ifferentiation
注意:经由L929骨髓细胞培养BMDM分化-conditioned介质收率〜98 %的CD11b + F4 / 80 + 巨噬细胞。


从细胞产量和FACS分析推导用于BMDM分化的L929条件培养基的最佳浓度
BMDM介质
加入15 %L929条件培养基以完成DMEM
在4°C下储存最多一个月
储备液的准备
对于Calcein -AM储备液,将50μg 固体Calcein -AM 溶解在50μl 无水DMSO中以制备1 mM储备液。将等分试样在-20°C的光线下冷冻在琥珀色反应管中长达4个月。避免多次冷冻/解冻
对于FeCl 2 储备液,将FeCl 2 溶解在超纯的优质水中,并在-20°C的温度下冻结1 mM储备液
对于8-羟基喹啉储备液,将8-羟基喹啉溶解在无水DMSO中,通过用PB S 稀释制备200μM 储备液,并将等分试样储存在-20°C
钙黄绿素-AM染色溶液
稀1 微升的1个mM的钙黄绿素-AM库存(FC 1 μM )的1ml PBS,涡旋并存储在黑暗中的溶液直到使用
立即在新鲜制备的Calcein -AM染色溶液中孵育BMDM,以确保最大的BMDM染色效率
FeHQ 解决方案
5添加微升1 毫的FeCl 2 (FC 。5 μM )至945 微升PBS和涡流
随后,添加50 微升的200 μM 8 hydroxyqu inoline(FC 10 μM )和涡流
在进行BMDM处理之前,每10分钟将FeHQ 溶液混合一次,以确保充分形成Fe 2+ 离子和8-羟基喹啉的络合物
胰蛋白酶溶液
添加500 微升10×胰蛋白酶至4.5毫升的PBS和1 00 微升500 毫摩尔EDTA(pH值= 8.0)
注意:对于一般的胰蛋白酶消化,至关重要的是使用不含Ca 2+ 和Mg 2+的PBS。


将胰蛋白酶溶液在37°C的水浴中孵育直至使用
F ACS缓冲区
溶解0. 5克BSA(FC 1 在50ml PBS%)
过滤灭菌并在4°C下将FACS 缓冲液存储最多1个月
 


致谢


 


该协议得到了奥地利科学基金项目InnateFun (FWF-I3319-B22)的支持,部分得到了EC-FP7计划向KK的项目FUNGITECT(HEALTH-F3-2013-602125)的支持。我们要感谢Du 等人的研究。(2015年)和Siegert 等人。(2015)建立这种优雅的方法。


 


利益争夺


 


作者宣称不存在利益冲突。


 


参考文献


 


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引用:Riedelberger, M. and Kuchler, K. (2020). Analyzing the Quenchable Iron Pool in Murine Macrophages by Flow Cytometry. Bio-protocol 10(6): e3552. DOI: 10.21769/BioProtoc.3552.
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