In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC₅₀, CC₅₀ and SI Values

Materials and Reagents

1. Cell culture flasks with filter cap, 25 cm² (Thermo Fisher Scientific, catalog number: 156367)
2. Cell culture flasks, plug seal cap, 25 cm² (Greiner Bio-One, catalog number: 690160)
3. 96-well flat bottom tissue culture plates (Sarstedt, catalog number: 83.3924.005)
4. Cell scrapers (Sarstedt, catalog number: 83.1830)
5. Cover glasses square (VWR, catalog number: 6311570)
6. Parafilm (Bemis, catalog number: HS234526B)
7. Pipette tips: 0.5-10 µl, 10-200 µl, 200-1,000 µl (Greiner Bio-One, catalog numbers: 771291, 739290, 740290)
8. Multichannel pipette reservoir (Brand, catalog number: BR703411)
9. Pleated filter paper (Sigma-Aldrich, catalog number: WHA1201150)
10. Microcentrifuge tubes, 1.5 ml (Greiner Bio-One, catalog number: 616201)
11. Serological pipettes 2 ml, 5 ml, 10 ml (Sarstedt, catalog numbers: 86.1252.001, 86.1253.001, 86.1254.001)
12. Syringes 5 ml (BD Emerald, catalog number: 307732)
13. Sterile syringe filter 0.22 µm (Millipore, catalog number: SLGVV255F) 
14. Leishmania infantum promastigotes (zymodeme GH8, strain MHOM/GR/2001/GH8) 
15. Leishmania major promastigotes (zymodeme LV39, strain MRHO/SU/59/P)
16. Immortalized macrophage cell line J774A.1 (ATCC; Rockville, USA/ ATCC No: TIB-67)
17. Total Phenolic Fraction (TPF) (derived from extra virgin olive oil from agricultural cooperative in Zaros region, Crete, Greece)
18. RPMI 1640 w/o L-glutamine (Biowest, catalog number: L0501)
19. Schneider’s insect medium (Biosera, catalog number: LM-F0702)
20. L-glutamine (Biosera, catalog number: LM-R1641)
21. HEPES buffer 1 M (Biowest, catalog number: L0180)
22. Penicillin-Streptomycin solution 10,000 U/ml (Biowest, catalog number: L0022)
23. Dimethyl sulfoxide (DMSO) cell culture grade (PanReac Applichem, catalog number: A3672,0050)
24. Ethanol absolute (Sigma-Aldrich, catalog number: 32205-M)
25. Formalin (Sigma-Aldrich, catalog number: R04586-82)
26. Milteforan^® 20 mg/ml (Hexadecylphosphocholine, [HePC], Virbac S.A.)
27. Resazurin sodium salt (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) (Sigma-Aldrich, catalog number: R7017)
28. Trypan blue dye for vital staining (BDH, catalog number: 34078)
29. Potassium chloride (KCl), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 746336)
30. Potassium phosphate monobasic (KH₂PO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795488)
31. Sodium chloride 99.9% (NaCl) (Applichem, catalog number: 381659)
32. Sodium phosphate dibasic (Na₂HPO₄), ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: 795410)
33. Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509)
34. Fetal Bovine Serum (Biowest, catalog number: S181B) (see Recipes)
35. Complete RPMI-1640 medium (see Recipes)
36. Complete Schneider’s medium (see Recipes)
37. Phosphate buffered saline, 1x (PBS, pH 7.2) (see Recipes)
38. Resazurin solution (see Recipes)
39. 0.4% (w/v) Trypan blue exclusion dye (see Recipes)
40. 0.01% w/v Sodium Dodecyl Sulfate (SDS) (see Recipes)
41. Lysis solution (see Recipes)
    

Equipment

1. Agitating water bath (Labtech, catalog number: LSB-015S)
2. ELISA Microplate reader (Dynatech Laboratories, catalog number: MRX)
3. Gilson pipettes (Gilson, models: PIPETMAN Classic P-10, P-20, P-200, P-1000)
4. Multichannel pipette (Brand, catalog number: 703710)
5. Malassez counting chamber (Paul Marienfeld GmbH & Co., catalog number: 0640610)
6. Microplate rotor (Centurion Scientific Ltd, catalog number: BRK 5530)
7. New Brunswick^TM Galaxy^® 170 S CO₂ Incubator (Eppendorf, catalog number: Galaxy 170 S)
8. Refrigerated centrifuge (Centurion Scientific Ltd, catalog number: PrO-Research K241R) 
9. Optical microscope (Olympus, catalog number: BHB)
10. pH meter (Thermo Fisher Scientific, catalog number: 13-644-928)
11. Pipette controller (Brand, catalog number: accu-jet^® pro 26300) 
12. Refrigerated Incubator 26 °C (Sanyo, catalog number: MIR-253)
13. Sterile biosafety cabinet (Telstar, catalog number: Bio-II-A) 
14. Water distiller (Sartorius, catalog number: H2O-I-1-UV-T)
    

Software

1. Microsoft^® Office Excel 2010 (Microsoft)

Procedure

1. In vitro cell culture method for biological evaluation of antileishmanial activity of total phenolic fraction (TPF) against Leishmania spp. promastigotes by using the resazurin reduction assay
   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 using the 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 (Koutsoni et al., 2018).
   2. Cultivate L. infantum and L. major promastigotes in 25 cm² cell culture flasks containing 10 ml of complete RPMI-1640 medium (Recipe 2) at 26 °C.
      Note: Long-term in vitro cultivation of Leishmania spp. leads to a progressive loss of virulence (Segovia et al., 1992). Subsequently, the maintenance of Leishmania spp. virulence is achieved by continuous passage in BALB/c mice. Tissue amastigotes are obtained after homogenization either of spleen tissue in case of L. infantum or popliteal lymph node in case of L. major. Transformation of intracellular amastigotes to infective promastigotes is achieved during culture in complete RPMI-1640 medium at 26 °C. Parasite cultures are subcultured into fresh medium (usually at day 4 depending on Leishmania strain, after every passage) until they reach the 10^th subculture (Nasiri et al., 2013). The appropriate inoculum dose of Leishmania spp. promastigotes is usually 2 x 10⁶ parasites per ml.
   3. Allow parasites to enter stationary-growth phase at 26 °C. Then promastigotes are removed by gently pipetting using a sterile disposable transfer pipette.
      Note: 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 it reaches the number of 3.5 x 10⁷ parasites/ml (Nasiri et al., 2013). 
   4. Determine the number of Leishmania spp. promastigotes per ml by differential counting of dead and live parasites using the Trypan blue exclusion dye (Recipe 6) in a Malassez counting chamber under an optical microscope.
      Note: 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^rth day of the in vitro culture. Additionally, promastigotes have to be fixed with 2% (v/v) formalin before counting. 
   5. Seed 1 x 10⁶ L. infantum promastigotes and 7.4 x 10⁵ L. major promastigotes per well in a final volume of 100 µl of complete RPMI-1640 medium in separate 96-well flat bottom tissue culture plate for each strain, under a sterile biosafety cabinet.
      Note: Different concentrations of stationary phase L. infantum and L. major promastigotes (e.g., 10⁴, 2.5 x 10⁴, 5 x 10⁴, 7.5 x 10⁴, 10⁵, 2.5 x 10⁵, 5 x 10⁵, 7.5 x 10⁵, 10⁶ in a final volume of 200 µl/well) are usually screened before the experiment’s set-up in order to determine the optimal cell density (Corral et al., 2013).
   6. 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.
   7. 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 3.3 µg/ml and 6.4 µg/ml for L. infantum and L. major promastigotes respectively, as previously described (Koutsoni et al., 2018).
   8. Triplicates of different kinds of negative controls are also included. More specifically, Leishmania spp. promastigotes cultured only in the presence of complete RPMI-1640 medium and Leishmania spp. promastigotes cultured in equivalent volumes of TPF’s solvents are included in triplicates (Figure 1).
      Note: A quadruplicate of parasite-free complete RPMI-1640 medium is also included in order to serve as blank.
      
      
      Figure 1. Example test plate designed to estimate IC₅₀ of TPF extract for Leishmania spp. promastigotes
      
      
   9. Seal the plates with parafilm and incubate them in a non-inverted position, in a 26 °C incubator for 60 h.
   10. Add resazurin solution (Recipe 5) at a final concentration of 20 µg/ml in each well and mix thoroughly by pipetting.
   11. Seal the plates with parafilm and further incubate them in a non-inverted position, in a 26 °C incubator until a fluorescent red color is observed in the negative control group (Figure 2).
       Note: Resazurin is a non-toxic, cell-permeable compound that converts from a non-fluorescent blue dye to the highly fluorescent red dye resorufin in response to changes of the reducing environment within the cytosol of the cell (Ahmed et al., 1994; Nociari et al., 1998). Noticeably, the incubation period depends on various parameters such as the parasite strain, varying from 3 to 72 h (Mikus and Steverding, 2000; Bilbao-Ramos et al., 2012; Corral et al., 2013; Kyriazis et al., 2013).
       
       
       Figure 2. Representative non-fluorescent blue wells (numbered 1-4) and fluorescent red wells (numbered 7-12)
       
       
   12. Read the plates by using an absorbance microplate reader with excitation at 570 nm and reference filter at 630 nm.
       Note: The excitation and emission spectra of resorufin are fairly broad, excitation filters between 530 and 570 nm can be used (Czekanska, 2011).
       
       
       
2. In vitro resazurin reduction assay to assess the concentration of TPF that results in 50% cytotoxicity (CC₅₀) in cultured macrophages 
   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 using the 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 at 37 °C under 5% CO₂ humidified air.
      Note: The J774A.1 macrophages used in experiments are long-term maintained in liquid nitrogen. More specifically, the J774A.1 macrophage cell line is freezed in complete RPMI-1640 medium supplemented with 10% DMSO and stored in liquid nitrogen in appropriate cryovials. For thawing procedure, cryovials are immediately placed in a 37 °C water bath and upon thawing, are transferred into a centrifugation tube containing pre-warmed complete growth medium. Cell suspension is centrifuged at 300 x g for 5 min. Gently, cells are resuspended in complete RPMI-1640 medium and transferred into 25 cm² cell culture flasks with filter cap at 37 °C under 5% 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 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 (see Video 1) (References 23 and 29)
      
      
      
      Video 1. Scraping
      
      
   5. 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.
      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^rth day of the in vitro culture. 
   6. Seed 4 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 a non-inverted position, in order to achieve cell adhesion.
   8. 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.
   9. Add one reference drug in triplicate at the appropriate 50% inhibitory concentration (IC₅₀). Hexadecylphosphocholine (HePC) is used at the concentrations of 60.2 µg/ml, as previously described (Koutsoni et al., 2018).
   10. Triplicates of different kinds of negative controls are also included. More specifically, J774A.1 macrophages cultured only in the presence of complete RPMI-1640 medium at a final volume of 200 µl and J774A.1 macrophages cultured in equivalent volumes of TPF’s solvents are included.
       Note: A quadruplicate of macrophages-free complete RPMI-1640 medium is also included in order to serve as blank. 
   11. Incubate the plate in a non-inverted position, at 37 °C under 5% CO₂ humidified air for 72 h.
   12. Add resazurin solution at a final concentration of 20 µg/ml in each well and mix thoroughly by pipetting.
   13. Incubate the plate in a non-inverted position, at 37 °C under 5% CO₂ humidified air until a fluorescent red color is observed in negative control group.
       Note: The incubation period of J774A.1 macrophages cultured only in the presence of complete RPMI-1640 medium, until the development of red color is usually 24 h.
   14. Read the plate by using an absorbance microplate reader with excitation at 570 nm and reference filter at 630 nm.
       
       
       
3. In vitro cell culture method for biological evaluation of antileishmanial activity of TPF extract against intracellular Leishmania spp. amastigotes by using the resazurin reduction assay
   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 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 with a cell scraper at 45° angle (see Video 1) (References 23 and 29).
   5. 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.
      Note: See the note of Step B5 in Procedure section. 
   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 in a non-inverted position, for 18 h at 37 °C under 5% CO₂ humidified air in order to achieve cell adhesion.
   8. 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.
      Note: Control triplicates of promastigote-free macrophages are also included in order to serve as infection negative control.
   9. Incubate the plate in a non-inverted position, 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-warmed at 37 °C with the use of a multichannel pipette at 45° angle.
   11. 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.
   12. Add one reference drug 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).
   13. Triplicates of different kinds of negative controls are also included. More specifically, Leishmania-infected J774A.1 macrophages cultured only in the presence of complete RPMI-1640 medium without drug influence and Leishmania-infected J774A.1 macrophages cultured in equivalent volumes of TPF’s solvents are included (Figure 3).
       Note: A quadruplicate of parasite- and cell-free complete RPMI-1640 medium is also included in order to serve as blank.
       
       
       Figure 3. Example test plate designed to estimate IC₅₀ of TPF extract for Leishmania spp. amastigotes
       
       
   14. Incubate the plate in a non-inverted position, for 48 h at 37 °C under 5% CO₂ humidified air.
   15. Remove culture supernatants with a multichannel pipette and disrupt macrophages’ membranes by adding 50 µl of lysis solution (Recipe 8) (Bilbao-Ramos et al., 2012).
   16. Incubate the plate in a non-inverted position, for 20 min at room temperature.
   17. Centrifuge the plate at 300 x g for 5 min in order to remove the cell lysis solution.
   18. Remove supernatant and replace by 200 µl/well of complete Schneider’s insect medium (Recipe 3).
       Note: Schneider’s insect medium is preferred as it is a more enriched in amino acids medium compared to RPMI-1640 medium and it is considered as a more effective medium in primary isolation from Leishmania spp. lesions (Grekov et al., 2011). 
   19. Seal the plate with parafilm and further incubate in a non-inverted position, in a 26 °C incubator for 72 h in order to allow transformation of viable amastigotes into promastigotes and proliferation.
   20. Add resazurin solution at a final concentration of 60 µg/ml in each well and mix thoroughly by pipetting.
   21. Seal the plate with parafilm and further incubate in a non-inverted position, at 26 °C until a fluorescent red color is observed in the negative control group (approximately for 24 h).
   22. Read the plate by using an absorbance microplate reader with excitation at 570 nm and reference filter at 630 nm. 

Data analysis

Resazurin assays offer a simple and rapid measurement for cell viability. Living cells are metabolically active and are able to reduce via mitochondrial reductase, the non-fluorescent dye resazurin to the strongly red-fluorescent dye resorufin (Rezende et al., 2019). The fluorescence output is proportional to the number of viable cells over a wide concentration range (O'Brien et al., 2000).

1. Data analysis for the estimation of 50% inhibitory concentration (IC₅₀) for Leishmania spp. promastigotes
   The correlation between promastigote density and the Optical Density (OD) acquired values is assessed by linear regression analysis with estimation of the coefficient of determination (R²) (Schneider et al., 2010).
   1. Calculate the average of the OD values acquired from the quadruplicate of parasite-free complete RPMI-1640 medium that serves as blank.
   2. Subtract this average value from every other OD value.
   3. Calculate the average OD values from every triplicate.
   4. The average OD value acquired from the triplicate of Leishmania spp. promastigotes cultured only in the presence of complete RPMI-1640 medium which serves as negative control, represents the 100% of parasite survival and growth and therefore a 0% of inhibition.
   5. Calculate the % of growth and correspondingly the % of inhibition for the various increasing concentrations of TPF, based on the average that refers to 100% of growth.
   6. Select all the values and insert a scatter plot that depicts the TPF concentrations in the x-axis and the % of inhibition in the y-axis.
      Note: The values on the y-axis are depicted as real numbers between 0 and 1.
   7. Set the x-axis to logarithmic scale and final graph must be approximately linear over some range.
      Note: The linear regression method of calculating the IC₅₀ value builts on the basis of the fact that all the data points for which the calculated percent of inhibition is more than 0% and less than 100%. This method assumes a linear relationship in the entire dose-response curve, which is rarely the case as it typically has a sigmoidal shape. To employ this method for proper IC₅₀ calculation, it usually has to be combined with the logarithmic transformation of one or both axes to ensure the proper conversion of the dose-response curve into the linear approximation (Nevozhay, 2014).
   8. Select “add trendline” and pick “linear” trendline options.
      Note: A trendline is most reliable when its R-squared value is at or near 1.
   9. Using the linear (y = ax + b) equation on this graph for y = 0.5 value, the x point equals to the IC₅₀ value (Figure 4). The IC₅₀ values are expressed in µg/ml.
      
      
      Figure 4. IC₅₀ calculation in Excel
      
      
      
2. Data analysis for the estimation of 50% cytotoxic concentration (CC₅₀) for J774A.1 macrophages
   The results are expressed as CC₅₀ value which is defined as the concentration of a compound that kills half of the cells in an uninfected cell culture (Smee et al., 2017).
   1. Calculate the average of the OD values acquired from the quadruplicate of macrophages-free complete RPMI-1640 medium that serves as blank.
   2. Subtract this average value from every other OD value.
   3. Calculate the average OD values from every triplicate.
   4. The average OD value acquired from the triplicate of J774A.1 macrophages cultured only in the presence of complete RPMI-1640 medium which serves as negative control, represents the 100% of macrophage growth and therefore a 0% of inhibition.
   5. Calculate the % of growth and correspondingly the % of inhibition for the various increasing concentrations of TPF, based on the average that refers to 100% of growth.
   6. Select all the values and insert a scatter plot that depicts the TPF concentrations in the x-axis and the % of inhibition in the y-axis.
      Note: The values on the y-axis are depicted as real numbers between 0 and 1.
   7. Set the x-axis to logarithmic scale and final graph must be approximately linear over some range.
      Note: The linear regression method of calculating the CC₅₀ value builts on the basis of the fact that all the data points for which the calculated percent of inhibition is more than 0% and less than 100%. This method assumes a linear relationship in the entire dose-response curve, which is rarely the case as it typically has a sigmoidal shape. To employ this method for proper CC₅₀ calculation, it usually has to be combined with the logarithmic transformation of one or both axes to ensure the proper conversion of the dose-response curve into the linear approximation (Nevozhay, 2014).
   8. Select “add trendline” and pick “linear” trendline options.
      Note: A trendline is most reliable when its R-squared value is at or near 1.
   9. Using the linear (y = ax + b) equation on this graph for y = 0.5 value, the x point equals to the CC₅₀ value. The CC₅₀ value is expressed in µg/ml.
      
      
      
3. Data analysis for the estimation of 50% inhibitory concentration (IC₅₀) for Leishmania spp. amastigotes
   1. Calculate the average of the OD values acquired from the quadruplicate of parasite- and macrophage-free complete RPMI-1640 medium that serves as blank.
   2. Subtract this average value from every other OD value.
   3. Calculate the average of the OD values acquired from the triplicate of promastigotes-free macrophages that serves as infection negative control.
      
   4. Subtract this average value from every other OD value.
   5. Calculate the average OD values from every triplicate.
   6. The average OD value acquired from the triplicate of Leishmania-infected J774A.1 macrophage cultured only in the presence of complete RPMI-1640 medium without any drug influence which serves as negative control, represents the 100% of parasite survival and growth and therefore a 0% of inhibition.
   7. Calculate the % of growth and correspondingly the % of inhibition for the various increasing concentrations of TPF, based on the average that refers to 100% of growth.
   8. Select all the values and insert a scatter plot that depicts the TPF concentrations in the x-axis and the % of inhibition in the y-axis.
      Note: The values on the y-axis are depicted as real numbers between 0 and 1.
   9. Set the x-axis to logarithmic scale and final graph must be approximately linear over some range.
      Note: The linear regression method of calculating the IC₅₀ value builts on the basis of the fact that all the data points for which the calculated percent of inhibition is more than 0% and less than 100%. This method assumes a linear relationship in the entire dose-response curve, which is rarely the case as it typically has a sigmoidal shape. To employ this method for proper IC₅₀ calculation, it usually has to be combined with the logarithmic transformation of one or both axes to ensure the proper conversion of the dose-response curve into the linear approximation (Nevozhay, 2014).
   10. Select “add trendline” and pick “linear” trendline options.
       Note: A trendline is most reliable when its R-squared value is at or near 1.
   11. Using the linear (y = ax + b) equation on this graph for y = 0.5 value, the x point equals to the IC₅₀ value. The IC₅₀ values are expressed in µg/ml.
       
       
       
4. Determination of the Selectivity Index (SI)
   The Selectivity Index (SI) is defined as the ratio of the 50% cytotoxic concentration of J774A.1 macrophages to the 50% inhibitory concentration of Leishmania spp. amastigotes (CC₅₀/IC₅₀). The in vitro activity of a tested compound against intracellular Leishmania spp. amastigotes in the absence of obvious cytotoxicity on murine macrophage cells (SI > 1) demonstrates its potential in the treatment of leishmaniasis (Makwali et al., 2015).

Notes

It is recommended to optimize experimental parameters, such as incubation time, the number of cells and parasites and the amount of resazurin used, before performing the assay for the first time since these parameters affect the consistency of the metabolic assay (Czekanska, 2011; Kim and Jang, 2018). Moreover, resazurin and resorufin are light-sensitive and need to be protected from light otherwise it results in decreased sensitivity.

Recipes

1. Fetal Bovine Serum (FBS)
   
   1. Thaw FBS and heat-inactivate it at 56 °C for 30 min in an agitating water-bath under constant agitation 
   2. Store at -20 °C until use
2. 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 of RPMI-1640 medium
   2. Store at 4 °C
   3. Take the appropriate volume and add FBS to 10% v/v final concentration
3. Complete Schneider’s medium
   
   1. Add 5 ml of Penicillin-Streptomycin (stock solution 10,000 U/ml stored at -20 °C)
   2. Store at 4 °C
   3. Take the appropriate volume and add FBS to 20% v/v final concentration
4. 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
   2. 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
   3. Add sterile and distilled water until final volume is 1,000 ml and sterilize through a 0.22 µm pore size syringe filter unit
   4. Prepare the 1x working solution by diluting 50 ml of 10x stock solution in 450 ml of sterile and distilled water
   5. Store at 4 °C
5. Resazurin solution
   
   1. Dissolve the appropriate amount of resazurin sodium salt in the desired final volume of PBS 1x (e.g., 2.46 mg of resazurin in 1 ml of PBS 1x to achieve an intermediate solution and then dilute 5 µl of this per well in order to achieve a final concentration of 60 µg/ml)
   2. Protect from light during experimental procedure
   3. Resazurin solution at working concentration must be prepared freshly each time before applying (Czekanska, 2011)
6. 0.4% (w/v) Trypan blue exclusion dye
   a.Dissolve 0.4 g of Trypan blue in 100 ml of PBS 1x. Agitate well
   b.Filter through a pleated filter paper and sterilize through a 0.22 µm pore size syringe filter unit
   c.Store at room temperature 
7. 0.01% (w/v) Sodium Dodecyl Sulfate (SDS)
   a. Dilute 0.01 g of SDS in 100 ml distilled water. Agitate well
   b. Store at room temperature
8. Lysis solution
   Dilute 4.8 µl of HEPES (stock solution 1 M) and 6 ml of 0.01 % SDS solution in RPMI-1640 medium in order to achieve a final volume of 10 ml
   

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 PhD 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 from Koutsoni et al., 2018 and Kyriazis et al., 2013.

Competing interests

Authors declare that they have no competing interests.

References

1. Ahmed, S. A., Gogal, R. M., Jr. and Walsh, J. E. (1994). A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [³H]thymidine incorporation assay. J Immunol Methods 170(2): 211-224.
2. Aykul, S. and Martinez-Hackert, E. (2016). Determination of half-maximal inhibitory concentration using biosensor-based protein interaction analysis. Anal Biochem 508: 97-103.
3. Bates, P. A. and Rogers, M. E. (2004). New insights into the developmental biology and transmission mechanisms of Leishmania. Curr Mol Med 4(6): 601-609.
4. Bilbao-Ramos, P., Sifontes-Rodriguez, S., Dea-Ayuela, M. A. and Bolas-Fernandez, F. (2012). A fluorometric method for evaluation of pharmacological activity against intracellular Leishmania amastigotes. J Microbiol Methods 89(1): 8-11.
5. Burza, S., Croft, S. L. and Boelaert, M. (2018). Leishmaniasis. Lancet 392(10151): 951-970.
6. Cheuka, P. M., Mayoka, G., Mutai, P. and Chibale, K. (2016). The role of natural products in drug discovery and development against neglected tropical diseases. Molecules 22(1): e58.
7. Corral, M. J., Gonzalez, E., Cuquerella, M. and Alunda, J. M. (2013). Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue. J Microbiol Methods 94(2): 111-116.
8. Czekanska, E. M. (2011). Assessment of cell proliferation with resazurin-based fluorescent dye. Methods Mol Biol 740: 27-32.
9. Grekov, I., Svobodova, M., Nohynkova, E. and Lipoldova, M. (2011). Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium. J Microbiol Methods 87(3): 273-277.
10. Griffiths, M. and Sundaram, H. (2011). Drug design and testing: profiling of antiproliferative agents for cancer therapy using a cell-based methyl-[³H]-thymidine incorporation assay. Methods Mol Biol 731: 451-465.
11. Kim, H. J. and Jang, S. (2018). Optimization of a resazurin-based microplate assay for large-scale compound screenings against Klebsiella pneumoniae. 3 Biotech 8(1): 3.
12. Koutsoni, O. S., Karampetsou, K., Kyriazis, I. D., Stathopoulos, P., Aligiannis, N., Halabalaki, M., Skaltsounis, L. A. and Dotsika, E. (2018). Evaluation of total phenolic fraction derived from extra virgin olive oil for its antileishmanial activity. Phytomedicine 47: 143-150.
13. Kyriazis, J. D., Aligiannis, N., Polychronopoulos, P., Skaltsounis, A. L. and Dotsika, E. (2013). Leishmanicidal activity assessment of olive tree extracts. Phytomedicine 20(3-4): 275-281.
14. Lage, O. M., Ramos, M. C., Calisto, R., Almeida, E., Vasconcelos, V. and Vicente, F. (2018). Current screening methodologies in drug discovery for selected human diseases. Mar Drugs 16(8): e279.
15. Lahlou, M. (2007). Screening of natural products for drug discovery. Expert Opin Drug Discov 2(5): 697-705.
16. Makwali, J. A., Wanjala, F. M. E., Ingonga, J. and Anjili, C. O. (2015). In vitro studies on the antileishmanial activity of herbicides and plant extracts against Leishmania major parasites. Res J Med Plant 9(3): 90-104.
17. Mikus, J. and Steverding, D. (2000). A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48(3): 265-269.
18. Mitra, A. K. and Mawson, A. R. (2017). Neglected tropical diseases: epidemiology and global burden. Trop Med Infect Dis 2(3): e36.
19. Nasiri, V., Karimi, G., Dalimi, A., Paykari, H. and Ghaffarifar, F. (2013). Effects of sheep and mouse urine on the growth pattern of Leishmania major promastigotes. Biomed Res Int 2013: 748592.
20. Nevozhay, D. (2014). Cheburator software for automatically calculating drug inhibitory concentrations from in vitro screening assays. PLoS One 9(9): e106186.
21. Nociari, M. M., Shalev, A., Benias, P. and Russo, C. (1998). A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods 213(2): 157-167.
22. O'Brien, J., Wilson, I., Orton, T. and Pognan, F. (2000). Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267(17): 5421-5426.
23. Passaging Cells- RAW.swf. Cannon Lab: You Tube.
24. Rezende, N., Ceron Jayme, C., Brassesco, M. S., Claudio Tedesco, A. and de Oliveira, H. F. (2019). Standardization of a resazurin-based assay for the evaluation of metabolic activity in oral squamous carcinoma and glioblastoma cells. Photodiagnosis Photodyn Ther 26: 371-374.
25. Schneider, A., Hommel, G. and Blettner, M. (2010). Linear regression analysis: part 14 of a series on evaluation of scientific publications. Dtsch Arztebl Int 107(44): 776-782.
26. Segovia, M., Artero, J. M., Mellado, E. and Chance, M. L. (1992). Effects of long-term in vitro cultivation on the virulence of cloned lines of Leishmania major promastigotes. Ann Trop Med Parasitol 86(4): 347-354.
27. Singh, N., Mishra, B. B., Bajpai, S., Singh, R. K. and Tiwari, V. K. (2014). Natural product based leads to fight against leishmaniasis. Bioorg Med Chem 22(1): 18-45.
28. Smee, D. F., Hurst, B. L., Evans, W. J., Clyde, N., Wright, S., Peterson, C., Jung, K. H. and Day, C. W. (2017). Evaluation of cell viability dyes in antiviral assays with RNA viruses that exhibit different cytopathogenic properties. J Virol Methods 246: 51-57.
29. TPP Cell Scrapers (www.midsci.com). MIDSCI Biotech: You Tube.