Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells

Materials and Reagents

1. 48-well plates, non-treated (CytoOne, STARLAB GmbH, catalog number: CC7672-7548 )
2. 48-well plates, tissue culture (tc) treated (greiner cellstar plate, Sigma-Aldrich, catalog number: M8937-100EA )
3. 96-well plate (U-shape, LIFE Technologies, catalog number: N8010560 )
4. Jurkat E6.1 T cells (Schneider et al., 1977, CVCL_0367); cultured in RPMI/10% FCS [v/v]
5. Mono Mac 6, MM6 (Ziegler-Heitbrock et al., 1988, CVCL_1426); Dectin-1-overexpressing MM6 cells were generated previously (Bode et al., 2019); cultured in RPMI1640/10% FCS/1% OPI [v/v]
6. Bone marrow-derived dendritic cells (BMDCs); See Bode et al. (2019) for further information BMDC generation
7. Bovine extracted catalase (Carl Roth, catalog number: 6025.1 ), storage at -20 °C, powder shelf-life ≥ 6 months, in solution only 1 day
8. Trolox (Th. Geyer, catalog number: CAY10011659-250 ), storage at 4 °C, powder shelf-life ≥ 6 months, in solution only ≤ 3 weeks
9. Dimethyl sulfoxide, sterile-filtered (BioPerformance Certified, Sigma-Aldrich, catalog number: D2438 )
10. sgp91 ds-tat, scrambled (Anaspc, catalog number: AS-63821 ), storage at -20 °C, shelf-life ≥ 24 months, avoid repeated freeze and thaw cycles
11. gp91 ds-tat (Anaspc, catalog number: AS-63818 ), storage at -20 °C, shelf-life ≥ 24 months, avoid repeated freeze and thaw cycles
12. GSK2795039 (MedChem Express, catalog number: HY-18950 ), storage at -20 °C, shelf-life ≥ 3 months, avoid repeated freeze and thaw cycles
13. RPMI 1640 (Sigma-Aldrich, catalog number: R8758-500ML )
14. Fetal calf serum, FCS (Sigma-Aldrich, catalog number: 12133C-500ML ), storage at -20 °C, shelf-life ≥ 12 months
15. OPI-supplement (Sigma-Aldrich, catalog number: O5003 ), storage at -20 °C, shelf-life ≥ 12 months
16. Polymyxin B sulfate salt (Sigma-Aldrich, catalog number: P4932 ), storage at 4 °C, powder shelf-life ≥ 12 months, in solution only ≤ 2 weeks
17. H₂DCFDA (Sigma-Aldrich, catalog number: D6883 ), storage at -20 °C, shelf-life ≥ 24 months, avoid repeated freeze and thaw cycles
18. N-acetyl cysteine, NAC (Sigma-Aldrich, catalog number: A7250 ), storage at 4 °C, shelf-life ≥ 6 months but in solution only 1 day
19. Phorbol-12-myristat-13-acetate (PMA; Sigma-Aldrich, catalog number: P8139-1MG ), storage at -20 °C, shelf-life ≥ 6 months but in solution only 1 day, avoid repeated freeze and thaw cycles
20. Depleted zymosan (DZ; Invivogen, catalog number: tlrl-zyd ), storage in solution at -20 °C, shelf-life ≥ 12 months, avoid repeated freeze and thaw cyclesGibco^TM PBS, pH 7.4 (Thermo Fisher Scientific, catalog number: 11503387 )
21. 500 mM H₂DCFDA solution (see Recipes)
22. 100 mM Trolox solution (see Recipes)
23. 100 mM NAC solution (see Recipes)
24. 1 mM Trolox stop solution (see Recipes)
25. 1 mM NAC stop solution (see Recipes)
26. Catalase mixture (ca. 10,000 U/ml) (see Recipes)
27. gp91 ds-tat, scrambled solution (see Recipes)
28. gp91 ds-tat solution (see Recipes)
29. GSK2795039 solution (see Recipes)
    

Equipment

1. Incubator (Heracell VIOS 160i, Thermo Fisher Scientific or similar product with the adjustment of 37 °C and 5% CO₂)
2. Centrifuge (Thermo Scientific Multifuge X1R Multi-Application, Thermo Fisher Scientific or similar product with 96-well inlay)
3. Single channel pipette, 2-20 µl (Gilson, catalog number: F144056M ; or similar product), use appropriate standard pipette tips (sterilized)
4. Single channel pipette, 20-200 µl (Gilson, catalog number: F144058M ; or similar product), use appropriate standard pipette tips (sterilized)
5. 8-channel pipette, 30-300 µl (Eppendorf Research, catalog number: 3125000052 ; or similar product), use appropriate standard pipette tips (sterilized)
6. Flow cytometer (FACSCanto II, BD Biosciences)
   

Software

1. Graph Pad Prism, version 6 (PrismGraphPad Software Inc., SCR_002798)
2. FlowJo, version 7.6.5 (BD Biosciences, SCR_008520)
   

Procedure

1. Prepare stock solutions and reaction mixtures (sterile conditions)
   
   
2. Plate cells (sterile conditions) (see also representative pipetting scheme in Figure 1)
   1. Use non-treated 48-well plates for MM6 and Jurkat T cells and tc-treated plates for adherent cells such as BMDCs and primary monocytes.
   2. Plate 100,000 cells/well (inner 24 wells) in 140 µl RPMI1640/10% FCS by using a 200 µl single channel pipette.
   3. Incubate at 37 °C for 30 min.
      
      
      Figure 1. Representative pipette scheme. Fill the outer wells with approximately 200 µl medium (gray-filled circles) to prevent evaporation of sample wells. Plate the cells in the 24 inner wells (open circles). Add medium or the appropriate control to the 12 wells on the left and add the appropriate ROS inhibitor to the 12 wells on the right (separated by the dotted line). Add medium or appropriate reference control (-), a positive control such as Phorbol-12-myristat-13-acetate (PMA) or depleted zymosan (DZ) (+), and the ROS-inducer of interest (X) in quadruplicates (or at least in triplicates).
      
      
      
3. Add ROS-inhibitor or appropriate control to the cells (sterile conditions) (see also representative pipetting scheme in Figure 1 and detailed information of the ROS-inhibitor in Table 1)
   
   Table 1. Specifications of the used ROS-inhibitors
   
   
   
   1. Prepare ROS-inhibitor dilutions in RPMI1640/10% FCS
      1. Catalase: at least 2,000 U/ml final conc.; Reference control: RPMI1640/10% FCS.
      2. Trolox: 5 mM final conc.; Reference control: equal volume of DMSO.
      3. NAC: 5 mM final conc.; Reference control: RPMI1640/10% FCS.
      4. gp91 ds-tat: at least 10 µM final conc.; Reference control: gp91 ds-tat scrambled.
      5. GSK2795039: 1-4 µM final conc.; Reference control: equal volume of DMSO.
   2. Place the plate with the seeded cells on a pre-warmed (37 °C) metal underlayment.
   3. Add 10 µl inhibitor or control solution by using a 20 µl single channel pipette.
   4. Place plate in cell culture incubator for 30 min at 37 °C.
   
   
4. Add ROS-inducing treatment or appropriate control to the cells (sterile conditions) (see Figure 1)
   1. Prepare treatment solutions such as PMA [100 ng/ml] in RPMI/10% FCS.
   2. Place the plate with the seeded cells on a pre-warmed (37 °C) metal underlayment.
   3. Add 50 µl treatment solution by using a 200 µl single channel pipette and incubate for 0.5-3 h in a cell culture incubator at 37 °C (the optimal incubation time depends on the individual treatments and needs to be empirically determined (e.g., by a kinetic approach).
      
      
      
5. For the last 30 min of incubation add H₂DCFDA solution to the cells (sterile conditions)
   1. Prepare H₂DCFDA solution in RPMI/10% FCS.
   2. Place the plate with the seeded cells on a pre-warmed (37 °C) metal underlayment.
      
      
      
6. Add 10 µl H₂DCFDA solution (5 mM final concentration) by using a 20 µl single channel pipette and incubate exactly for 30 min in a cell culture incubator at 37 °C.
   
   
7. Finally, add Trolox or NAC solution to cells to terminate the reaction (sterile conditions)
   
   1. Place the plate with the seeded cells on a pre-warmed (37 °C) metal underlayment.
   2. Add 50 µl stop solution by using a 200 µl single channel pipette and incubate for 10 min at 37 °C.
   
   
8. Prepare cells for analysis
   1. Place the plate with the seeded cells at 4 °C in the dark for 10 min to slow down cell metabolism (during this step fluorescently-labeled antibodies against specific cell surface markers may be added to discriminate subpopulations; do not use FITC- or Alexa488-labeled antibodies).
   2. Carefully resuspend the cells by using a 200 µl single channel pipette (3-4 times) and transfer cells into a 96-well plate (U-shape).
   3. Spin down the cells at 300 x g for 5 min at 4 °C.
   4. Quickly remove supernatants.
   5. Resuspend cells in 300 µl ice-cold Trolox or NAC stop solution by using a 300 µl 8-channel pipette.
      
      
      
9. Analyze cells via flow cytometry within the FITC-channel (see also Figure 2)
   1. Keep cells on ice in the dark.
   2. Gate on living cells by morphology (FITC-positive events represent living and metabolically active cells).
   3. Exclude cell doublets by using the forward scatter (FSC)-A and FSC-H.
   4. Perform the measurement as soon as possible within 1 h after adding the stop solution.
      
      
      Figure 2. Gating strategy and data analysis of intracellular ROS detection experiments using H₂DCFDA. A. ROS production in cells leads to the conversion of H₂DCFDA into a green fluorescent compound which can be detected within the FITC-channel by flow cytometry. Gate on living cells (determined by morphology and/or by gating on FITC positive events; not shown) and exclude cell doublets by using the forward scatter (FSC)-A and FSC-H. B. Representative histograms depicting the ROS-induction in Dectin-1-overexpressing MM6 cells mediated by recombinant Annexin A1 (blue) compared to untreated (grey) in non-pre-treated MM6 cells (left histogram) or catalase pre-treated MM6 cells (right histogram; Ziegler-Heitbrock et al., 1988). C. Quantification of one representative experiment measured in triplicates. The mean ± S.D. of the FITC-mean fluorescent intensity (MFI) is depicted. D. Statistical analysis of six independent experiments. The mean ± S.D. of the relative FITC-MFI increase in % of non-pre-treated MM6 cells compared to catalase pre-treated cells is depicted (MFI treated – MFI untreated)/MFI untreated * 100). This subfigure was taken from Bode et al., 2019. ***P < 0.001 (paired two-tailed t-test). E. Representative ROS-induction by 100 ng/ml Phorbol-12-myristat-13-acetate (PMA) and 40 µg/ml depleted Zymosan (DZ) in Dectin-1-overexpressing MM6 cells using the NOX-2-specific peptide inhibitor gp91 ds-tat or the control peptide (Ctrl.) quantified as in panel D. The mean ± S.E.M. of triplicates (DZ) or quadruplicates (PMA) is shown.

Data analysis

Data analysisIntracellular ROS induce conversion of H₂DCFDA into a cell-impermeable green fluorescent product which can be quantified by flow cytometry within the FITC-channel (Ex: 488 nm/Em: 519 nm) as mean/median fluorescent intensity (MFI). To determine the ROS signal induced by the treatment, the MFI should be normalized to untreated (or control) cells ((MFI treated–MFI untreated)/MFI untreated * 100). For every independent experiment, use at least triplicates for each condition. For statistical analysis perform at least 3 independent experiments and summarize the specific FITC-MFI increases in % relative to untreated or control treated cells as described above (see Figure 2) and use the paired two-tailed t-test to determine significant differences in ROS-production.

Notes

1. Add the H₂DCFDA in the same order to the wells as the treatments were added.
2. The use of freshly prepared Trolox and NAC solutions is strongly recommended.
3. Using over night air oxidized RPMI1640/10% FCS may enhance the sensitivity of the measurement.
4. Handle not more than 48 wells/reactions (2 plates) at the same time to ensure short assay times.
   

Recipes

1. 500 mM H₂DCFDA solution
   in DMSO
   Storage at -20 °C; shelf-life ≤ 24 months, avoid repeated freeze and thaw cycles
2. 100 mM Trolox solution
   in DMSO
   Storage at 4 °C; shelf-life ≤ 3 months
3. 100 mM NAC solution
   in PBS
   Adjust pH to 7.4
   Storage at 4 °C; shelf-life 1 day
4. 1 mM Trolox stop solution
   in RPMI1640/10% FCS [v/v]
   Storage at 4 °C; shelf-life 1 day
5. 1 mM NAC stop solution
   in RPMI1640/10% FCS [v/v]
   Storage at 4 °C; shelf-life 1 day
6. Catalase mixture (ca. 10,000 U/ml)
   in RPMI1640/10 % FCS [v/v]
   Storage at 4 °C; shelf-life 1 day; do not use a metallic spoon for transfer the catalase powder
7. gp91 ds-tat
   According to the manufacturer’s instruction
   Storage at -20 °C, shelf-life ≥ 24 months, avoid repeated freeze and thaw cycles
8. gp91 ds-tat, scrambled solution
   Scrambled according to the manufacturer’s instruction
   Storage at -20 °C, shelf-life ≥ 24 months, avoid repeated freeze and thaw cycles
9. GSK2795039 solution
   According to the manufacturer’s instruction, storage at -20 °C, shelf-life ≥ 3 months, avoid repeated freeze and thaw cycles
   

Acknowledgments

We thank J. Steiner, R. Mölle, T. Machauer, M. Trapp, V. Neubauer and T. Vornbäumen (all German Cancer Research Center, Heidelberg, Germany) for technical support. We also thank K. Gülow (University Hospital Regensburg, Germany) for critical discussions. Moreover, we thank H. Sauter for excellent secretarial assistance. We also thank V. Heil for proofreading the manuscript. This work was supported by the German Federal Ministry of Education and Research VIP+ validation fund. This protocol was adapted from previous work of Bode and colleagues (Bode et al., 2019).

Competing interests

The authors declare no conflict of financial or scientific interests.

Ethics

The generation of BMDCs was approved by the veterinary authorities (Regierungspräsidium Karlsruhe) of Baden-Württemberg (DKFZ346).

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