Materials and Reagents

Cell Culture Flasks with filter cap, 25 cm² (Thermo Fisher Scientific, catalog number: 156367)
Cell culture Flasks, plug seal cap, 25 cm² (Greiner Bio-One, catalog number: 690160)
Cell scrapers (Sarstedt, catalog number: 83.1830)
Cover glasses square (VWR, catalog number: 6311570)
Falcon^® Round-Bottom Tubes, 5 ml (Corning, catalog number: 352008)
96-well flat bottom tissue culture plates (Sarstedt, catalog number: 83.3924.005)
24-well flat bottom tissue culture plates (Sarstedt, catalog number: 83.3922.005)
Pipettes tips: 0.5-10 μl, 10-200 μl, 200-1,000 μl (Greiner Bio-One, catalog numbers: 771291, 739290, 740290)
Pleated filter paper (Sigma-Aldrich, catalog number: WHA1201150)
Reaction tubes, 1.5 ml (Greiner Bio-One, catalog number: 616201)
Serological pipettes 2 ml, 5 ml, 10 ml (Sarstedt, catalog numbers: 86.1252.001, 86.1253.001, 86.1254.001)
Sterile syringe filter 0.22 μm (Millipore, catalog number: SLGVV255F)
Syringes 5 ml (Terumo, catalog number: SS+05S21381)
Cell lifter (Corning, catalog number: 3008)
Leishmania infantum promastigotes (zymodeme GH8, strain MHOM/GR/2001/GH8)
Leishmania major promastigotes (zymodeme LV39, strain MRHO/SU/59/P)
Immortalized macrophage cell line J774A.1 (ATCC; Rockville, USA/ ATCC No: TIB-67)
Total Phenolic Fraction (derived from Extra Virgin Olive Oil from agricultural cooperative in Zaros region, Crete, Greece)
RPMI-1640 (w/o) L-glutamine (Biowest, catalog number: L0501)
Fetal Bovine Serum (Biowest, catalog number: S181B)
L-glutamine (Biosera, catalog number: LM-R1641)
HEPES buffer 1 M (Biowest, catalog number: L0180)
Penicillin-Streptomycin solution 10,000 U/ml (Biowest, catalog number: L0022)
Dimethyl sulfoxide (DMSO) Cell culture grade (PanReac Applichem, catalog number: A3672,0050)
Ethanol absolute (Sigma-Aldrich, catalog number: 32205-M)
Formalin (Sigma-Aldrich, catalog number: R04586-82)
Trypan blue dye for vital staining (BDH, catalog number: 34078)
Milteforan^® 20 mg/ml (Hexadecylphosphocholine, [HePC], Virbac S.A.)
Hydrochloric acid (HCl) solution, 1 M (Sigma-Aldrich, catalog number: 150696)
Sodium chloride 99.9% (NaCl) (Applichem, catalog number: 381659)
Potassium chloride (KCl), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 746336)
Sodium phosphate dibasic (Na₂HPO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795410)
Potassium phosphate monobasic (KH₂PO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795488)
Sodium hydroxide (NaOH) solution, 1 M (Sigma-Aldrich, catalog number: 79724)
Carboxy-H₂DCFDA (Thermo Fisher Scientific, catalog number: C400)
Hydrogen Peroxide solution 30% (w/w) (Sigma-Aldrich, catalog number: H1009)
ortho-Phosphoric acid 85% (AppliChem, catalog number: A0989)
Sulfanilamide (Sigma-Aldrich, catalog number: S9251-100G)
N-(1-Naphthyl) ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488)
Sodium nitrite (NaNO₂) (Sigma-Aldrich, catalog number: 237213)
Complete RPMI-1640 medium (see Recipes)
Fetal Bovine Serum solution (FBS) (see Recipes)
Phosphate Buffer Saline (PBS), 10x, pH 7.2-7.4 (see Recipes)
0.4% (w/v) Trypan blue exclusion dye (see Recipes)
Sodium nitrite (NaNO₂) solution (see Recipes)
Griess reagent (see Recipes)
H₂O₂ solution (see Recipes)

Equipment

New Brunswick^TM Galaxy^® 170 S CO₂ Incubator (Eppendorf, catalog number: Galaxy 170 S)
Refrigerated Incubator 26 °C (Sanyo, catalog number: MIR-253)
BD FACSCalibur Flow Cytometer: 3-Color (BD FACSCalibur^TM, catalog number: 342973)
ELISA Microplate reader (Dynatech Laboratories, catalog number: MRX)
Optical microscope (Olympus, catalog number: BHB)
Sterile biosafety cabinet (Telstar, catalog number: Bio-II-A)
Gilson pipettes (Gilson, catalog numbers: PIPETMAN Classic P-10, P-20, P-200, P-1000)
Malassez counting chamber (Paul Marienfeld GmbH & Co., catalog number: 0640610)
Multichannel pipette (Brand, catalog number: 703710)
pH Meter (Thermo Fisher Scientific, catalog number: 13-644-928)
Pipette controller (Brand, catalog number: accu-jet^® pro 26300)
Water distiller (Sartorius, catalog number: H2O-I-1-UV-T)

Software

Microsoft^® Office Excel 2010 (Microsoft)
FlowJo, version 10 (BD)

Procedure

In vitro quantification of nitric oxide production by Leishmania-infected J774A.1 macrophage treated with increasing concentrations of TPF
1. Prepare a known concentration of the plant extract using the appropriate solvent. TPF is dissolved in 62.5% pure ethanol, 31.25% sterile distilled water and 6.25% DMSO.
  Note: The recovery of TPF from extra virgin olive oil was carried out by Centrifugal Partition Extraction (CPE) technique, which is an innovative solid support free separation technique derived from Centrifugal Partition Chromatography (CPC). Briefly, liquid-liquid chromatography was performed using a laboratory scale centrifugal partition extractor FCPE300^®, which was equipped with a rotor composed of 7 stacked partition disks engraved with a total of 231 partition cells, while the total volume of the column was 300 ml (Angelis et al., 2017; Koutsoni et al., 2018).
2. Cultivate J774A.1 macrophage in 25 cm² cell culture flasks with filter cap containing 10 ml of complete RPMI-1640 medium (Recipe 1) at 37 °C under 5% CO₂ humidified air.
3. Allow macrophages to reach a high density population of about 70% confluence.
  Note: J774A.1 macrophages cultured in complete RPMI-1640 medium at 37 °C under 5% CO₂ humidified air, usually reach 70% confluence within 3 to 4 days.
4. Then, remove the majority of culture medium with a sterile disposable transfer pipette and leave about 2 ml medium in the flask and detach macrophage monolayer by scrapping cells gently and slowly with a cell scraper at 45° angle.
5. Determine the number of macrophages per ml by differential counting of dead and live cells using the Trypan blue exclusion dye (Recipe 4) in a Malassez counting chamber under an optical microscope.
  Note: The final dilution of macrophages in Trypan blue dye depends on the density of the culture. Usually, a 1:20 final dilution of macrophages is suitable at the 3^rd or 4^th day of the in vitro culture.
6. Seed 5 x 10⁴ J774A.1 macrophages per well in a final volume of 100 μl of complete RPMI-1640 medium in a 96-well flat bottom tissue culture plate under a sterile biosafety cabinet.
7. Incubate the plate for 18 h at 37 °C under 5% CO₂ humidified air in order to achieve cell adhesion.
8. Determine the number of Leishmania spp. promastigotes per ml (promastigotes must be in their stationary phase of growth) by differential counting of dead and live parasites using the Trypan blue exclusion dye in a Malassez counting chamber under an optical microscope.
  Notes:
  1. Leishmania spp. promastigotes usually enter the stationary-growth phase after 3 to 5 days, depending on the Leishmania strain. For example, L. major, a commonly used strain, approximately enters the stationary growth phase at Day 4 when reaches the number of 3.5 x 10⁷ parasites/ml (Nasiri et al., 2013).
  2. The final dilution of promastigotes in Trypan blue dye depends on the density of the in vitro parasite culture. Usually, a 1:20 final dilution of promastigotes is suitable at the 3^rd or 4^th day of the in vitro culture. Additionally, promastigotes have to be fixed with 2% v/v formalin before counting.
9. Seed 7.5 x 10⁵ Leishmania spp. early stationary phase promastigotes in each well (i.e., ratio of 15:1 parasites/macrophage) in a total final volume of 200 μl of complete RPMI-1640 medium.
10. Incubate the plate for 48 h at 37 °C under 5% CO₂ humidified air.
11. Then, remove the non-internalized promastigotes by washing thrice with RPMI-1640 medium pre-warmed at 37 °C.
12. Add various increasing concentrations of TPF (5-400 μg/ml) in triplicates and fill in the wells with complete RPMI-1640 medium until the final volume of 200 μl per well.
  Note: Concentrations of plant extracts may vary depending on the natural product.
13. Add one reference drug (i.e., one drug currently subscribed for the chemotherapy of leishmaniasis) in triplicate at the appropriate 50% inhibitory concentration (IC₅₀). Hexadecylphosphocholine (HePC) is used at the concentrations of 0.6 μg/ml and 3.2 μg/ml for L. infantum and L. major respectively, as previously described (Koutsoni et al., 2018).
  Note: Control triplicates of the equivalent volumes of TPF solvents must also be included.
14. Triplicate of a negative control group must also be included. More specifically, a triplicate of Leishmania-infected J774A.1 macrophages cultured only in the presence of complete RPMI-1640 medium without any drug influence, serves as a negative control group.
15. Incubate the plate for 48 h at 37 °C under 5% CO₂ humidified air.
16. Collect culture supernatants and transfer 50 μl of each sample into a new 96-well flat bottom tissue culture plate, as designated in Figure 1. It is also recommended to use again triplicates for each sample.
  Note: Culture supernatants contain the secreted reactive nitrogen intermediates.
  
  Figure 1. Representative illustration of the 96-well flat bottom plate that Griess assay takes place
17. A sodium nitrite standard curve is required. Place 100 μl of NaNO₂ solution (Recipe 5) at concentration of 75 μg/ml at A1 and A2 wells, as designated in Figure 1.
18. Place 50 μl of distilled H₂O in B1, B2 etc. until H1 and H2 wells and perform serial dilutions by transferring 50 μl of NaNO₂ solution from A1 and A2 to B1 and B2 respectively until H1 and H2, with the use of a multichannel pipette.
  Note: Discard the excessive 50 μl obtained from H1 and H2 wells.
19. Place 100 μl of Griess reagent (Recipe 6) in both standards and samples subsequently.
  Notes:
  1. Griess reagent must be freshly prepared. When preparing Griess reagent, avoid contact with the bottleneck because this usually contains Griess-positive material that gives an increase in background absorbance.
  2. The final standard NaNO₂ concentrations range from 0.195 μg/ml to 25 μg/ml.
  3. A quadruplicate that contains only 50 μl of distilled water that serves as blank is also included.
20. Read the plate by using an absorbance microplate reader with excitation at 570 nm.
  Note: Color change from transparent to pink is immediately noticed. The intensity of the color is greater as nitrite concentration gets higher.
In vitro quantification of reactive oxygen species production by Leishmania-infected J774A.1 macrophages treated with increasing concentrations of TPF
1. Prepare a known concentration of the plant extract using the appropriate solvent. TPF is dissolved in 62.5% pure ethanol, 31.25% sterile distilled water and 6.25% DMSO.
  Note: The recovery of TPF from extra virgin olive oil was carried out by Centrifugal Partition Extraction (CPE) technique, which is an innovative solid support free separation technique derived from Centrifugal Partition Chromatography (CPC), as mentioned in Procedure section, Step A1.
2. Cultivate J774A.1 macrophages in 25 cm² cell culture flasks with filter cap, containing 10 ml of complete RPMI-1640 medium, as previously described in Step A2.
3. Allow macrophages to reach a high density population of about 70% confluence, as mentioned in Step A3.
4. Then, determine the number of macrophages per ml by differential counting of dead and live cells using the Trypan blue exclusion dye in a Malassez counting chamber under an optical microscope, as already described in Steps A4 and A5.
5. Seed 5 x 10⁵ J774A.1 macrophages per well in a final volume of 500 μl of complete RPMI-1640 medium in a 24-well flat bottom tissue culture plate under a sterile biosafety cabinet (Figure 2).
  
  Figure 2. Representation of the 24-well tissue culture plate
6. Incubate the plate for 18 h at 37 °C under 5% CO₂ humidified air in order to achieve cell adhesion.
7. Determine the number of Leishmania spp. promastigotes per ml (promastigotes must be in their stationary phase of growth) by differential counting of dead and live parasites using the Trypan blue exclusion dye in a Malassez counting chamber under an optical microscope.
  Notes:
  1. Leishmania spp. promastigotes usually enter the stationary-growth phase after 3 to 5 days, depending on the Leishmania strain. For example, L. major, approximately enters the stationary growth phase at Day 4 when reaches the number of 3.5 x 10⁷ parasites/ml (Nasiri et al., 2013).
  2. The final dilution of promastigotes in Trypan blue dye depends on the density of the in vitro parasite culture. Usually, a 1:20 final dilution of promastigotes is suitable at the 3^rd or 4^th day of the in vitro culture. Additionally, promastigotes have to be fixed with 2% v/v formalin before counting.
8. Seed 7.5 x 10⁶ Leishmania spp. early stationary phase promastigotes in each well (i.e., ratio of 15:1 parasites/macrophage) in a total final volume of 1,000 μl of complete RPMI-1640 medium.
  Note: Infection negative control group is included and is consisted of promastigote-free macrophages.
9. Incubate the plate for 48 h at 37 °C under 5% CO₂ humidified air.
10. Then, remove the non-internalized promastigotes by washing thrice with RPMI-1640 medium pre-heated at 37 °C.
11. Add various increasing concentrations of TPF (5-400 μg/ml) and fill in the wells with complete RPMI-1640 medium until the final volume of 1,000 μl per well.
  Note: Concentrations of plant extracts may vary depending on the natural product.
12. Add one reference drug (i.e., one drug currently subscribed for the chemotherapy of leishmaniasis) in triplicate at the appropriate 50% inhibitory concentration (IC₅₀). Hexadecylphosphocholine (HePC) is used at the concentrations of 0.6 μg/ml and 3.2 μg/ml for L. infantum and L. major respectively, as previously described (Koutsoni et al., 2018).
  Notes:
  1. Control of the equivalent volumes of TPF solvents must also be included.
  2. Three different control groups must be included i) Leishmania spp. infected-macrophages that will serve as treatment negative control, ii) Leishmania spp. infected-macrophages that will serve later as positive control for ROS production and iii) Leishmania spp. infected-macrophages that will serve later in the flow cytometry as the non-labeled sample.
13. Incubate the plate for 48 h at 37 °C under 5% CO₂ humidified air.
14. Remove culture supernatants and add 500 μl of PBS 1x solution (Recipe 3) in each well except from the positive control for ROS production where you add 500 μl of 1 mM H₂O₂ solution (Recipe 7).
  Note: H₂O₂ is an inducer of ROS production by the macrophages and therefore is useful as positive control for the ROS production (Ogawa et al., 2004).
15. Incubate the plate for 15 min at 37 °C under 5% CO₂ humidified air.
16. Remove the culture supernatant from the positive control group and replace with 500 μl of PBS 1x solution.
17. Add 5 μM of the carboxy-H₂DCFDA in each well.
  Note: In the non-labeled group, avoid adding the carboxy-H₂DCFDA.
18. Incubate the plate for 30 min at 37 °C under 5% CO₂ humidified air.
19. Remove the culture supernatants and add in each well 500 μl of PBS 1x solution.
20. Detach cells with the use of a cell lifter.
21. Collect one by one cell suspensions and transfer them into a 5 ml round bottom tube each.
  Note: Tubes containing cell suspensions should be placed on ice during the procedure of the flow cytometric data collection.
22. Analyze the samples in the FACSCalibur.

Data analysis

In vitro quantification of NO production by Leishmania-infected J774A.1 macrophages treated with increasing concentrations of TPF
Griess colorimetric nitrite assay relies on the conversion of the nitric oxide in a sample to a stable azo compound that absorbs at around 540 nm (Sun et al., 2003). Put the plate in the microplate reader in order to obtain the Optical Density (OD) value of each well.
1. Calculate the mean Optical Density (OD) value of the blank quadruplicate and subtract it from every other OD value.
2. Calculate the mean of the OD values for each of the standard nitrite concentrations.
3. Select all the values that correspond to the standard nitrite concentrations and insert a scatter plot that depicts the nitrite concentration in the x-axis and the mean of the corresponding OD values in the y-axis.
4. Extract the linear region of the curve.
5. Select “add trendline” and pick “linear” trendline options.
6. Add the trendline in the chart and extract the equation and R-squared value of the trendline (Figure 3.).
  Note: A trendline is most reliable when its R-squared value is at or near 1.
  
  Figure 3. Linear region of the standard curve with trendline
7. Calculate the mean of the OD values that corresponds to each sample.
8. Based on the equation, find the nitrite concentration for each sample.
In vitro quantification of ROS production by Leishmania-infected J774A.1 macrophages treated with increasing concentrations of TPF
For the data analysis, FlowJo software is used.
1. Analyze the samples according to their size (forward-scattered light/ FSC) and cell granularity (side-scattered light/ SSC) on a dual parameter dot plot and set a gating region to exclude cellular debris (Figure 4).
  Note: As a general guide, cellular debris is FSC-low.
  
  Figure 4. An example of gating J774A.1 macrophages analyzed by FSC and SSC dot plot
2. Analyze the gated cell population in a single-parameter histogram (FL1-H channel).
3. Extract the Geo-mean for each sample (Figure 5).
  Note: Geo-mean is representative of the fluorescence intensity of the sample. The greater the Geo-mean, the more is the fluorescence intensity and consequently the more is the presence of ROS in the cells (Eruslanov and Kusmartsev, 2010).
  
  Figure 5. Representative single-parameter histograms of flow-cytometric analysis for the quantification of ROS production. FL1-histogram of the fluorescence intensity (Geo Mean) in gated population of J774A.1 cells loaded with H₂DCFDA upon stimulation with (A) RPMI-1640 medium and (B) TPF at 400 µg/ml.

Recipes

Complete RPMI-1640 medium
1. Add 5 ml of L-glutamine (stock solution 200 mM, stored at -20 °C), 5 ml of Penicillin-Streptomycin (stock solution 10,000 U/ml, stored at -20 °C) and 5 ml of HEPES (stock solution 1 M) to 500 ml RPMI-1640 medium
2. Store at 4 °C
3. Take the appropriate volume and add FBS to 10% (v/v) final concentration
Fetal Bovine Serum solution (FBS)
Thaw FBS and heat-inactivate it at 56 °C for 30 min in a water-bath under constant agitation
Store at -20 °C until use
Phosphate Buffer Saline (PBS), 10x, pH 7.2-7.4
1. Place 800 ml of distilled water in a suitable container and dissolve 80 g NaCl, 2 g KCl, 11.5 g of Na₂HPO₄, and 2 g of KH₂PO₄. Agitate until the complete salt dilution and adjust solution to the desired pH (typically 7.2-7.4) with NaOH (1 M) or HCl (1 M) solution, if needed
2. Add distilled water until the final volume is 1,000 ml and sterilize through a 0.22 μm pore size syringe filter unit
3. Prepare the 1x working solution by diluting 50 ml of 10x stock solution in 450 ml of sterile and distilled water. Store at 4 °C
0.4% (w/v) Trypan blue exclusion dye
1. Dissolve 0.4 g of Trypan blue in 100 ml of PBS 1x. Agitate well
2. Filter through a pleated filter paper and sterilize through a 0.22 μm pore size syringe filter unit. Store at room temperature
Sodium nitrite (NaNO₂) solution
Prepare a stock solution of 75 µg/ml
Dissolve 750 µg in 10 ml of de-ionized water
Griess reagent
Griess reagent consists of two separate solutions (A and B) that they are mixed together (ratio 1:1) at the moment of the experiment operation
Griess reagent A
First, prepare a 3% (v/v) solution of phosphoric acid. Then, dissolve the appropriate amount of sulfanilamide at a final concentration of 1% (w/v)
Griess reagent B
Dissolve the appropriate amount of N-(1-Naphthyl) ethylenediamine dihydrochloride in distilled water in order to obtain a final concentration of 0.1 % (w/v)
H₂O₂ solution
1. Prepare 100 ml working solution of 1 mM
2. Dissolve 10 μl of stock solution (30% w/w) in 100 ml of de-ionized water

Acknowledgments

Part of this research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI Ph.D. Fellowship grant (GA. no. 6ΝΔΘ46ΨΖ2Ν-ΣΣΟ). Also, this work was supported by KRHPIS II (MIS 5002486) and EATRIS (MIS 5028091).
The protocols were adapted and modified by Koutsoni et al., 2018 and Kyriazis et al., 2016.

Competing interests

Authors declare that they have no competing interests.

References

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