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Aug 2018
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In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values
天然复合产物抗利什曼原虫活性的体外筛选:IC50、CC50和SI值的测定   

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Abstract

Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols for the in vitro screening of a natural product-derived compound required for the determination of its antileishmanial potency. For this purpose, the Total Phenolic Fraction (TPF) derived from extra virgin olive oil is evaluated through the in vitro cell culture method against extracellular promastigote and intracellular amastigote Leishmania spp. forms. The aim of this article is to describe a step-by-step procedure that can be easily applied to accurately estimate the 50% inhibitory concentration (IC50), the 50% cytotoxic concentration (CC50) and the selectivity index (SI) via the resazurin reduction assay. These protocols are based on the ability of resazurin (oxidized blue form) to be irreversibly reduced by enzymes in viable cells and generate a red fluorescent resorufin product and can be easily expanded to the investigation of the antimicrobial activity in other microorganisms.

Keywords: Cytotoxic concentration (细胞毒性浓度), Inhibitory concentration (抑制浓度), Selectivity index (选择性指数), Natural products (天然产物), Leishmania spp. (利什曼原虫属), Macrophages (巨噬细胞)

Background

Neglected tropical diseases (NTDs) are a group of about 20 diseases, typically endemic in 149 tropical and subtropical countries that represent a significant public health impact worldwide by affecting more than 1 billion people and being responsible for over 500,000 deaths per year (Cheuka et al., 2016; Mitra and Mawson, 2017). Until nowadays, drug discovery against NTDs exhibited limited success in translating potential drug candidates into effective therapies. Indeed, the current clinically used drugs against NTDs are characterized by various disadvantages including severe adverse effects, complicated administration procedures, lengthy treatment duration and emergence of resistance (Cheuka et al., 2016). Natural products represent a valuable alternative source of novel and structurally diverse compounds that deserves attention in drug discovery against NTDs. Numerous metabolites isolated from plant, microbial and marine sources such as alkaloids, phenolic compounds, quinones, terpenes, saponins, lignans, toxoids and anthranoids have been investigated (Cheuka et al., 2016) and it has been estimated that approximately at least 25% of the presently used drugs in modern medicine are derived from plants (Lahlou, 2007).

Among NTDs, leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania spp., causing a range of manifestations and exhibiting progressively increasing frequency in non-endemic western developed countries (Burza et al., 2018). Numerous of agents of natural origin, plant derived bioactive compounds and their secondary metabolites have been tested as antileishmanial drugs, such as flavonoids, sterols, chalcones, coumarins, tannins and aurones, iridoids, quinones and quinolone alkaloids (Singh et al., 2014). The assessment of the bioactive potential of biological extracts or molecules involves numerous assays performed at animal, cell-based or molecular levels (Lage et al., 2018) while the first steps of the biological screening and evaluation of plant extracts usually involve the screening of compounds in cell-based in vitro assays (Griffiths and Sundaram, 2011).

The half maximal inhibitory concentration (IC50) is a representative quantitative measure of the potency of a substance to inhibit in vitro a specific biological process by 50%, (Lahlou, 2007; Aykul and Martinez-Hackert, 2016) and is primarily defined through in vitro test models. Since Leishmania spp. have a digenetic life cycle, the antileishmanial effect of drugs is tested on both the promastigote (infective form in the intermediate host) and the amastigote form (intracellular form in the tissue of the mammalian host) (Bates and Rogers, 2004). In this regard, IC50 is determined for both promastigotes and intracellular amastigotes and is defined as the concentration of a compound that causes inhibition of growth in the 50% of Leishmania promastigotes and of intracellular amastigotes, respectively. To this end, the in vitro screening of the antileishmanial activity of Total Phenolic Fraction (TPF) is performed on L. infantum and L. major promastigotes and on Leishmania-infected J774A.1 macrophages in which amastigotes survive and multiply (Bilbao-Ramos et al., 2012). Moreover, the cytotoxicity of TPF in vitro against J774A.1 macrophages is also tested through the determination of the CC50 (cytotoxicity concentration 50%) that is the compound’s concentration that causes cytotoxicity in the 50% of the cells (Kyriazis et al., 2013). In addition, we employ the selectivity index (SI) that 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). A compound with SI value greater than 1 is considered to be more selective against Leishmania spp. parasites and is regarded as a promising potential agent in the treatment of leishmaniasis (Makwali et al., 2015). All the above indices are crucial in order to conclude whether or not a tested compound is appropriate as an antileishmanial agent.

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 BrunswickTM 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 10th 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 3rd or 4rth 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 3rd or 4rth 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

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  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.
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  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.
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  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.
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简介

被忽视的热带病获得了众多研究计划的科学兴趣,以期实现对其的有效控制或消除。在这种尝试中,需要更多前沿的公共卫生政策和研究来发现源自天然产物的新的,更安全和有效的药物。在这里,我们描述了用于体外筛选天然产物衍生化合物以确定其抗疟药效力所需的方案。为此,通过体外细胞培养方法对细胞外前鞭毛体和胞内鞭毛体利什曼原虫 spp评估了来自特级初榨橄榄油的总酚分数(TPF)。形式。本文的目的是描述一个循序渐进的过程,该过程可以轻松地应用于准确估算50%抑制浓度(IC 50 )和50%细胞毒性浓度(CC 50 )和通过刃天青素还原试验的选择性指数(SI)。这些协议基于刃天青(氧化蓝色形式)在活细胞中被酶不可逆地还原并产生红色荧光试卤灵产物的能力,并且可以轻松地扩展到研究其他微生物的抗菌活性。
【背景】被忽视的热带病(NTDs)大约由20种疾病组成,通常在149个热带和亚热带国家中流行,通过影响超过10亿人口并每年造成500,000多例死亡,在全世界范围内代表着重大的公共卫生影响(Cheuka et al。,2016; Mitra和Mawson,2017)。直到今天,针对NTD的药物发现在将潜在的候选药物转化为有效疗法方面仅显示出有限的成功。的确,当前针对NTDs的临床使用药物具有多种缺点,包括严重的副作用,复杂的给药程序,漫长的治疗时间和耐药性的出现(Cheuka et al。,2016)。天然产物是新型和结构多样的化合物的有价值的替代来源,在针对NTD的药物发现中值得关注。已经研究了从植物,微生物和海洋资源中分离出的许多代谢物,如生物碱,酚类化合物,醌,萜烯,皂苷,木脂素,类毒素和类蒽(Cheuka等人,2016年),并且已经进行了研究。据估计,现代医学中目前至少约25%的药物来自植物(Lahlou,2007年)。

在NTD中,利什曼病是由 Leishmania 属的原生动物引起的寄生虫病,在非地方性西方发达国家引起了一系列表现并呈逐渐增加的趋势(Burza et al。 ,2018年)。许多天然来源的药剂,植物来源的生物活性化合物及其次生代谢产物已被作为抗疟药进行了测试,例如黄酮类化合物,固醇,查耳酮,香豆素,丹宁酸和金质,虹彩化合物,醌和喹诺酮生物碱(Singh et al。 ,2014)。对生物提取物或分子的生物活性潜力的评估涉及在动物,基于细胞或分子水平上进行的大量测定(Lage et al。,2018),而生物学筛选和评估的第一步植物提取物通常涉及在基于细胞的体外试验中筛选化合物(Griffiths和Sundaram,2011年)。

最大半数抑制浓度(IC 50 )是一种物质在体外抑制特定生物过程50%的效力的代表性定量度量,(Lahlou,2007 ; Aykul和Martinez-Hackert,2016年),主要通过体外测试模型进行定义。自利什曼原虫 spp。由于具有双基因生命周期,在前鞭毛体(中间宿主中的感染形式)和鞭毛体形式(哺乳动物宿主组织中的细胞内形式)上都测试了药物的抗菌作用(Bates and Rogers,2004)。在这方面,测定前鞭毛体和胞内变形虫的IC 50 ,并将其定义为引起50%的 Leishmania 前鞭毛体和细胞内的变形虫分别。为此,在 L上进行体外筛选总酚醛成分(TPF)的抗霉菌活性。婴儿和 L。主要前鞭毛体和利什曼原虫感染的J774A.1巨噬细胞,其中无鞭毛体得以生存和繁殖(Bilbao-Ramos等人,2012)。此外,还通过测定CC 50 (细胞毒性浓度50%)来测试TPF 体外对J774A.1巨噬细胞的细胞毒性。 50%的细胞具有细胞毒性(Kyriazis et al。,2013)。此外,我们采用选择性指数(SI),其定义为J774A.1巨噬细胞的50%细胞毒性浓度与利什曼原虫 spp的50%抑制浓度之比。 amastigotes(CC 50 / IC 50 )。 SI值大于1的化合物被认为对利什曼原虫 spp更具选择性。寄生虫,被认为是治疗利什曼病的有希望的潜在药物(Makwali et al。,2015)。以上所有指标对于确定被测化合物是否适合作为抗霉菌剂至关重要。

关键字:细胞毒性浓度, 抑制浓度, 选择性指数, 天然产物, 利什曼原虫属, 巨噬细胞

材料和试剂

  1. 带滤帽的细胞培养瓶,25 cm 2 (Thermo Fisher Scientific,目录号:156367)
  2. 细胞培养瓶,塞子密封盖,25 cm 2 (Greiner Bio-One,目录号:690160)
  3. 96孔平底组织培养板(Sarstedt,目录号:83.3924.005)
  4. 细胞刮板(Sarstedt,目录号:83.1830)
  5. 方形玻璃罩(VWR,目录号:6311570)
  6. 封口膜(Bemis,目录号:HS234526B)
  7. 移液器吸头:0.5-10 µl,10-200 µl,200-1,000 µl(Greiner Bio-One,目录号:771291、739290、740290)
  8. 多通道移液器储液器(品牌,目录号:BR703411)
  9. 褶状滤纸(Sigma-Aldrich,目录号:WHA1201150)
  10. 1.5 ml微量离心管(Greiner Bio-One,目录号:616201)
  11. 血清移液管2 ml,5 ml,10 ml(Sarstedt,目录号:86.1252.001,86.1253.001,86.1254.001)
  12. 注射器5毫升(BD翡翠,目录号:307732)
  13. 无菌注射器过滤器0.22 µm(Millipore,目录号:SLGVV255F)
  14. 婴儿利什曼原虫前鞭毛体(zymodeme GH8,菌株MHOM / GR / 2001 / GH8)
  15. 大利什曼原虫前鞭毛体(zymodeme LV39,品系MRHO / SU / 59 / P)
  16. 永生化巨噬细胞系J774A.1(ATCC;美国罗克维尔/ ATCC编号:TIB-67)
  17. 总酚含量(TPF)(来自希腊克里特岛扎罗斯地区农业合作社的特级初榨橄榄油)
  18. 不含L-谷氨酰胺的RPMI 1640(Biowest,目录号:L0501)
  19. 施耐德的昆虫培养基(Biosera,目录号:LM-F0702)
  20. L-谷氨酰胺(Biosera,目录号:LM-R1641)
  21. HEPES缓冲液1 M(Biowest,目录号:L0180)
  22. 青霉素-链霉素溶液10,000 U / ml(Biowest,目录号:L0022)
  23. 二甲基亚砜(DMSO)细胞培养等级(PanReac Applichem,目录号:A3672,0050)
  24. 无水乙醇(Sigma-Aldrich,目录号:32205-M)
  25. 福尔马林(Sigma-Aldrich,目录号:R04586-82)
  26. Milteforan ® 20 mg / ml(十六烷基磷酸胆碱,[HePC],Virbac S.A.)
  27. 刃天青钠盐(7-羟基-3H-苯恶嗪-3-一10-氧化物)(Sigma-Aldrich,目录号:R7017)
  28. 台盼蓝染料用于重要染色(BDH,目录号:34078)
  29. 氯化钾(KCl),ACS试剂,≥99.0%(Sigma-Aldrich,目录号:746336)
  30. 磷酸二氢钾(KH 2 PO 4 ),ACS试剂,≥99.0%(Sigma-Aldrich,目录号:795488)
  31. 氯化钠99.9%(NaCl)(Applichem,目录号:381659)
  32. 磷酸氢二钠(Na 2 HPO 4 ),ACS试剂,≥99.0%(Sigma-Aldrich,目录号:795410)
  33. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
  34. 胎牛血清(Biowest,目录号:S181B)(请参阅食谱)
  35. 完整的RPMI-1640培养基(请参阅食谱)
  36. 填写施耐德的媒体(请参阅食谱)
  37. 磷酸盐缓冲盐水,1x(PBS,pH 7.2)(请参阅食谱)
  38. 刃天青溶液(参见配方)
  39. 0.4%(w / v)台盼蓝拒染染料(请参阅配方)
  40. 0.01%w / v十二烷基硫酸钠(SDS)(请参阅食谱)
  41. 裂解液(请参阅食谱)

设备

  1. 搅拌水浴(Labtech,目录号:LSB-015S)
  2. ELISA酶标仪(Dynatech Laboratories,目录号:MRX)
  3. 吉尔森移液器(吉尔森,型号:PIPETMAN Classic P-10,P-20,P-200,P-1000)
  4. 多通道移液器(品牌,目录号:703710)
  5. Malassez计数室(Paul Marienfeld GmbH& Co.,目录号:0640610)
  6. 微孔板转子(Centurion Scientific Ltd,目录号:BRK 5530)
  7. New Brunswick TM Galaxy ® 170 S CO 2 培养箱(Eppendorf,目录号:Galaxy 170 S)
  8. 冷藏离心机(Centurion Scientific Ltd,目录号:PrO-Research K241R)
  9. 光学显微镜(奥林巴斯,货号:BHB)
  10. pH计(Thermo Fisher Scientific,目录号:13-644-928)
  11. 移液器控制器(品牌,目录号:Accu-jet ® pro 26300)
  12. 冷藏培养箱26°C(Sanyo,目录号:MIR-253)
  13. 无菌生物安全柜(Telstar,目录号:Bio-II-A)
  14. 蒸馏水机(Sartorius,目录号:H2O-I-1-UV-T)

软件

  1. Microsoft ® Office Excel 2010(Microsoft)

程序

  1. 体外细胞培养方法对总酚类组分(TPF)抗利什曼原虫 spp的抗疟疾活性进行生物学评估。使用刃天青素还原试验测定前鞭毛体
    1. 使用适当的溶剂制备已知浓度的植物提取物。 TPF溶于62.5%的纯乙醇,31.25%的无菌蒸馏水和6.25%的DMSO。
      注:使用离心分离萃取(CPE)技术从特级初榨橄榄油中回收TPF,这是一种创新的无固体载体分离技术,源自离心分离色谱(CPC)。简而言之,使用实验室规模的离心隔板萃取器FCPE300 ® 进行液-液色谱,该离心机配备了由7个堆叠隔板组成的转子圆盘上刻有总共231个分配单元,而柱子的总体积为300毫升(Koutsoni 等人 ,2018)。
    2. 培养 L。婴儿和 L。在25 cm 2 细胞培养瓶中的主要前鞭毛体,在26°C下装有10 ml完全RPMI-1640培养基(配方2)。
      注意: 利什曼原虫 spp的长期 体外 培养。导致毒力的逐步丧失(Segovia 等人。 ,1992)。随后,维护 利什曼原虫 spp。通过在BALB / c小鼠中连续传代可实现毒力。在 L的情况下,将脾脏组织均质化后即可获得组织变形虫。婴儿 或淋巴结 L。主要 。在26°C的完全RPMI-1640培养基中培养期间,可实现胞内变形虫向感染性前鞭毛虫的转化。将寄生虫培养物传代培养到新鲜培养基中(通常在第4天,取决于传代后的 Leishmania 菌株),直到达到第10个 传代培养( Nasiri 等。 ,2013年)。 利什曼原虫 spp的合适接种剂量。前鞭毛体通常是每毫升2 x 10 6 寄生虫。
    3. 让寄生虫在26°C进入平稳生长阶段。然后使用无菌的一次性移液管轻轻移液除去前鞭毛体。
      注意: 利什曼原虫病 spp。前鞭毛体通常在3-5天后进入稳定生长阶段,具体取决于 利什曼原虫 菌株。例如 L。主要 ,一种常用菌株,当达到3.5 x 10 7 寄生虫/ ml的数量时(Nasiri et al。 ,2013)。
    4. 确定利什曼原虫 spp的数量。通过在光学显微镜下在Malassez计数室中使用锥虫蓝排除染料(配方6)对死活寄生虫进行死活活寄生虫的计数,可以测定每毫升前鞭毛体的含量。
      注意:前鞭毛虫在锥虫蓝染料中的最终稀释取决于 体外 寄生虫培养物的密度。通常,在体外 的第3天或第4天,将前鞭毛体最终稀释度为1:20是合适的em>文化。此外,计数前,前鞭毛体必须用2%(v / v)福尔马林固定。
    5. 种子1 x 10 6 L。婴儿前鞭毛体和7.4 x 10 5 L。在无菌生物安全柜中,每个菌株的每个孔中含有主要前鞭毛体,最终体积为100 µl完整RPMI-1640培养基,每个菌株分别置于96孔平底组织培养板中。
      注意:不同浓度的固定相 L。婴儿 和 L。主要 前鞭毛体(例如10 4 ,2.5 x 10 4 ,5 x 10 4 ,7.5 x 10 4 ,10 5 ,2.5 x 10 5 ,5 x 10 5 ,7.5 x 10 5 ,10 6 ,最终体积为200 µl /孔)通常在实验开始之前进行筛选,以确定最佳的细胞密度(Corral 等人。 ,2013)。
    6. 一式三份添加各种浓度递增的TPF(5-400 µg / ml),并用完整的RPMI-1640培养基填充孔中,直到最终体积为每孔200 µl。
      注意:植物提取物的浓度可能因天然产物而异。
    7. 以适当的50%抑制浓度(IC 50 )一式三份添加一种参考药物(即,一种目前正在参与利什曼病化学治疗的药物)。十六烷基磷酸胆碱(HePC)的浓度为3.3 µg / ml, L的浓度为6.4 µg / ml。婴儿和 L。如前所述(Koutsoni et al。,2018)。
    8. 一式三份的各种阴性对照也包括在内。更具体地说,是利什曼原虫 spp。前鞭毛体仅在完全RPMI-1640培养基和利什曼原虫 spp的存在下培养。在等体积的TPF溶剂中培养的前鞭毛体一式三份(图1)。
      注意:还包括一式四份的无寄生虫完全RPMI-1640培养基,以用作空白。


      图1.设计用于估算 利什曼病的TPF提取物的IC 50 的示例测试板 spp。前鞭毛体

    9. 用石蜡膜密封培养板,并在26°C的培养箱中以不可倒置的方式培养60 h。
    10. 在每个孔中添加终浓度为20 µg / ml的刃天青溶液(配方5),并通过移液彻底混合。
    11. 用石蜡膜密封板,然后在26°C的培养箱中以非反转的方式进一步孵育,直到在阴性对照组中观察到荧光红色(图2)。
      注意:刃天青是一种无毒的,可渗透细胞的化合物,响应细胞胞质溶胶中还原性环境的变化,它可以从无荧光的蓝色染料转变为高荧光的红色试卤灵(Ahmed 等。 ,1994; Nociari 等。 ,1998)。值得注意的是,潜伏期取决于各种参数,例如寄生虫菌株,范围从3到72 h不等(Mikus和Steverding,2000; Bilbao-Ramos 等人。 ,2012 ; Corral 等。 ,2013; Kyriazis 等。 ,2013)。


      图2.代表性的非荧光蓝色孔(编号1-4)和荧光红色孔(编号7-12)

    12. 通过使用酶标仪在570 nm处激发和630 nm处的参考滤光片读取板。
      注意:间苯二酚的激发和发射光谱相当宽,可以使用530至570 nm之间的激发滤光片(捷克,2011年)。

  2. 通过体外刃天青素还原试验来评估导致培养的巨噬细胞产生50%细胞毒性(CC 50 )的TPF浓度。
    1. 使用适当的溶剂制备已知浓度的植物提取物。 TPF溶于62.5%的纯乙醇,31.25%的无菌蒸馏水和6.25%的DMSO。
      注意:如步骤部分所述,使用离心分配萃取(CPE)技术从特级初榨橄榄油中回收TPF,这是一种创新的无固体支持物分离技术,源自离心分离色谱(CPC)。 ,步骤A1。
    2. 在25 cm 2 细胞培养瓶中培养J774A.1巨噬细胞,并在37°C和5%CO 2 湿润空气下,用装有10 ml完全RPMI-1640完全培养基的滤器盖。
      注意:实验中使用的J774A.1巨噬细胞可长期保存在液氮中。更具体地说,将J774A.1巨噬细胞细胞系在补充有10%DMSO的完全RPMI-1640培养基中冷冻,并储存在液氮中的适当冷冻管中。对于解冻过程,将冷冻管立即置于37°C水浴中,解冻后,将其转移至装有预热的完全生长培养基的离心管中。将细胞悬液在300 x g 离心5分钟。轻轻地将细胞重悬于完全的RPMI-1640培养基中,并转移到25°Ccm 2 带有过滤器盖的细胞培养瓶中,在37°C下5 %的加湿空气。
    3. 使巨噬细胞达到约70%汇合的高密度种群。
      注意:通常在5%CO 2 湿空气中,于37°C在RPMI-1640完全培养基中培养的J774A.1巨噬细胞,通常在3到4天内达到70%的汇合度。
    4. 然后,除去大部分培养基,并在烧瓶中保留约2 ml培养基,并通过用细胞刮刀以45°角轻轻和缓慢地刮擦细胞来分离巨噬细胞单层(请参阅视频1)(参考23和29)


      视频1.抓取

    5. 在光学显微镜下,使用Malpanz计数室中的锥虫蓝排除染料,通过对死活细胞进行差异计数来确定每毫升巨噬细胞的数量。
      注:巨噬细胞在台盼蓝染料中的最终稀释取决于培养物的密度。通常,在3 rd 或4 rth处最终稀释1:20的巨噬细胞是合适的 体外培养的一天。
    6. 在无菌生物安全柜下的96孔平底组织培养板中,每孔播种4 x 10 4 J774A.1巨噬细胞,最终体积为100 µl完整RPMI-1640培养基。
    7. 将平板在37°C,5%CO 2 湿润空气中于非反转位置孵育18小时,以实现细胞粘附。
    8. 一式三份添加各种浓度递增的TPF(5-400 µg / ml),并用完整的RPMI-1640培养基填充孔中,直到最终体积为每孔200 µl。
      注意:植物提取物的浓度可能因天然产物而异。
    9. 以适当的50%抑制浓度(IC 50 )一式三份加入一种参考药物。如前所述(Koutsoni等人,2018),十六烷基磷酸胆碱(HePC)的使用浓度为60.2μg/ ml。
    10. 一式三份的各种阴性对照也包括在内。更具体地说,包括仅在完全体积为200 µl的完整RPMI-1640培养基中培养的J774A.1巨噬细胞和在等体积的TPF溶剂中培养的J774A.1巨噬细胞。
      注意:还包含一式四份的无巨噬细胞的完整RPMI-1640培养基,以用作空白。
    11. 在37°C和5%CO 2 湿润空气下,将培养板在同相位置孵育72小时。
    12. 在每个孔中添加终浓度为20 µg / ml的刃天青溶液,并通过移液彻底混合。
    13. 在37°C的5%CO 2 湿润空气下,以非反转的方式孵育板,直到阴性对照组中观察到荧光红色。
      注意:仅在完全RPMI-1640培养基存在下培养J774A.1巨噬细胞的潜伏期,直到通常出现24小时出现红色为止。
    14. 通过使用酶标仪在570 nm处激发和630 nm处的参考滤光片读取酶标板。

  3. 体外细胞培养方法对TPF提取物对细胞内 Leishmania spp的抗猪鞭毛活性的生物学评估。刃天青还原法测定氨基苯甲酸酯
    1. 使用适当的溶剂制备已知浓度的植物提取物。 TPF溶于62.5%的纯乙醇,31.25%的无菌蒸馏水和6.25%的DMSO。
      注意:如步骤部分所述,采用离心分配萃取(CPE)技术从特级初榨橄榄油中回收TPF,这是一种创新的无固体载体分离技术,源自离心分离色谱(CPC),步骤A1。
    2. 在25 cm 2 细胞培养瓶中培养J774A.1巨噬细胞,该培养瓶盖中装有10 ml完整RPMI-1640培养基,在37°C和5%CO 2 湿润空气下进行。
    3. 使巨噬细胞达到约70%汇合的高密度种群。
      注意:通常在5%CO 2 湿空气中,于37°C在RPMI-1640完全培养基中培养的J774A.1巨噬细胞,通常在3到4天内达到70%的汇合度。
    4. 然后,用无菌的一次性移液管移走大部分培养基,并在烧瓶中保留约2 ml培养基,并通过用细胞刮刀以45°角轻轻刮擦细胞来分离巨噬细胞单层(参见视频1)(参考23和29) 。
    5. 在光学显微镜下,使用Malpanz计数室中的锥虫蓝排除染料,通过对死活细胞进行差异计数来确定每毫升巨噬细胞的数量。
      注意:请参阅“过程”部分中步骤B5的注释。
    6. 每孔中接种5 x 10 4 J774A.1巨噬细胞,在无菌生物安全柜下的96孔平底组织培养板中,将最终体积为100 µl完整RPMI-1640培养基接种。
    7.  在5%CO 2 湿润空气中于37°C于非反转位置孵育板18小时,以实现细胞粘附。
    8. 种子7.5 x 10 5 利什曼原虫 spp。每个孔中的早期静止期前鞭毛体(即,寄生虫与巨噬细胞的比例为15:1),最终最终体积为200 µl完全RPMI-1640培养基。
      注意:为了避免感染,阴性对照也包括一式三份不含前鞭毛体的巨噬细胞。
    9. 将培养板在37°C下,5%CO 2 湿润空气中以相同的方向孵育48小时。
    10. 然后,通过使用45°角的多通道移液器,在37°C下预热的RPMI-1640培养基洗涤三次,除去未内在化的前鞭毛体。
    11. 一式三份添加各种浓度递增的TPF(5-400 µg / ml),并用完整的RPMI-1640培养基填充孔中,直到最终体积为每孔200 µl。
      注意:植物提取物的浓度可能因天然产物而异。
    12. 以适当的50%抑制浓度(IC 50 )一式三份加入一种参考药物。十六烷基磷酸胆碱(HePC)的浓度为0.6 µg / ml, L的浓度为3.2 µg / ml。婴儿和 L。 ,如先前所述(Koutsoni 等人,,2018年)。
    13. 一式三份的各种阴性对照也包括在内。更具体地说,仅在没有药物影响的完全RPMI-1640培养基中培养被 Leishmania 感染的J774A.1巨噬细胞和以相同体积培养的被 Leishmania 感染的J774A.1巨噬细胞包括TPF的溶剂(图3)。
      注意:还包括一式四份无寄生虫和无细胞的完整RPMI-1640培养基,以用作空白。


      图3.设计用于估算 利什曼病的TPF提取物的IC 50 的示例测试板 spp。变形虫

    14. 将培养板在37°C下,5%CO 2 湿润空气中以相同的方向孵育48小时。
    15. 用多通道移液器除去培养上清液,并加入50 µl裂解液破坏巨噬细胞膜(第8条)(Bilbao-Ramos等人,2012年)。
    16. 在室温下,将培养板在非反转位置孵育20分钟。
    17. 将板在300 xem离心5分钟,以去除细胞裂解液。
    18. 除去上清液,并以每孔200 µl的完整施耐德昆虫培养基(配方3)替换。
      注意:首选施耐德昆虫培养基,因为它比RPMI-1640培养基富含氨基酸,并且被认为是从 利什曼原虫初次分离中更有效的培养基。 > spp。病变(Grekov 等。 ,2011年)。
    19. 用石蜡膜密封培养板,然后在26°C的培养箱中以非颠倒位置进一步培养72小时,以便将有活力的变形虫转化为前鞭毛体并增殖。
    20. 在每个孔中添加终浓度为60 µg / ml的刃天青溶液,并通过移液彻底混合。
    21. 用石蜡膜密封板,然后在26°C的非反转位置进一步孵育,直到在阴性对照组中观察到荧光红色(约24 h)。
    22. 通过使用在570 nm处激发和630 nm处的参考滤光片的吸光度酶标仪读取板。

数据分析

刃天青测定法提供了简单而快速的细胞活力测定。活细胞具有代谢活性,能够通过线粒体还原酶(非荧光染料刃天青)还原为强红色荧光染料试卤灵(Rezende et al。,2019)。荧光输出与在宽浓度范围内的活细胞数量成正比(O'Brien等人,2000)。

  1. 估计利什曼原虫 spp的50%抑制浓度(IC 50 )的数据分析。前鞭毛体
    通过线性回归分析和确定系数(R 2 )的估计,评估了前鞭毛体密度与光密度(OD)获得值之间的相关性(Schneider et al。 ,2010)。
    1. 计算从一式四份无寄生虫的完整RPMI-1640培养基(用作空白)中获得的OD值的平均值。
    2. 从其他OD值中减去该平均值。
    3. 计算每个一式三份的平均OD值。
    4. 从利什曼原虫 spp一式三份获得的平均OD值。仅在完全RPMI-1640培养基(作为阴性对照)存在下培养的前鞭毛体代表100%的寄生虫存活和生长,因此抑制率为0%。
    5. 基于表示100%增长的平均值,计算各种浓度的TPF的增长百分比以及相应的抑制百分比。
    6. 选择所有值并插入一个散点图,该点在x轴上描述TPF浓度,在y轴上描述抑制百分比。
      注意:y轴上的值表示为0到1之间的实数。
    7. 将x轴设置为对数刻度,并且最终图形必须在某个范围内近似线性。
      注意:计算IC 50 值的线性回归方法基于以下事实:计算出的抑制百分比大于0%且小于100%。该方法在整个剂量反应曲线中都假设为线性关系,这种情况很少见,因为它通常呈S型。为了将这种方法用于正确的IC 50 计算,通常必须将其与一个或两个轴的对数转换结合使用,以确保正确的IC剂量反应曲线转化为线性近似值(Nevozhay,2014年)。
    8. 选择“添加趋势线”并选择“线性”趋势线选项。
      注意:趋势线的R平方值等于或接近1时最可靠。
    9. 使用此图上的线性(y = ax + b)方程,对于y = 0.5值,x点等于IC 50 值(图4)。 IC 50 值以µg / ml表示。


      图4. IC 50 Excel中的计算

  2. 估计J774A.1巨噬细胞50%细胞毒性浓度(CC 50 )的数据分析
    结果表示为CC 50 值,该值定义为杀死未感染细胞培养物中一半细胞的化合物的浓度(Smee et al。,2017) 。
    1. 计算从无空巨噬细胞的完整RPMI-1640培养基(一式四份)的一式四份中获得的OD值的平均值。
    2. 从其他OD值中减去该平均值。
    3. 计算每个一式三份的平均OD值。
    4. 从仅在存在完整RPMI-1640培养基(作为阴性对照)的情况下培养的J774A.1巨噬细胞一式三份获得的平均OD值代表了100%的巨噬细胞生长,因此抑制率为0%。
    5. 基于表示100%增长的平均值,计算各种浓度的TPF的增长百分比以及相应的抑制百分比。
    6. 选择所有值并插入一个散点图,该点在x轴上描述TPF浓度,在y轴上描述抑制百分比。
      注意:y轴上的值表示为0到1之间的实数。
    7. 将x轴设置为对数刻度,并且最终图形必须在某个范围内近似线性。
      注意:计算CC 50 值的线性回归方法基于以下事实:计算出的抑制百分比大于0%且小于100%。该方法在整个剂量反应曲线中都假设为线性关系,这种情况很少见,因为它通常呈S型。为了将这种方法用于正确的CC 50 计算,通常必须将其与一个或两个轴的对数转换结合使用,以确保正确的CC将剂量反应曲线转化为线性近似值(Nevozhay,2014年)。
    8. 选择“添加趋势线”并选择“线性”趋势线选项。
      注意:趋势线的R平方值等于或接近1时最可靠。
    9. 对于此y = 0.5值,使用此图上的线性(y = ax + b)方程,x点等于CC 50 值。 CC 50 值以µg / ml表示。

  3. 估计利什曼原虫 spp的50%抑制浓度(IC 50 )的数据分析。变形虫
    1. 计算从无空白和无巨噬细胞的完整RPMI-1640完整培养基(一式四份)中获得的OD值的平均值。
    2. 从其他OD值中减去该平均值。
    3. 计算从无前鞭毛体的巨噬细胞一式三份获得的OD值的平均值,该巨噬细胞用作感染阴性对照。
    4. 从其他OD值中减去该平均值。
    5. 计算每个一式三份的平均OD值。
    6. 仅在完全RPMI-1640培养基(无任何药物影响)作为阴性对照的情况下培养的,由三次被Leemmania 感染的J774A.1巨噬细胞一式三份获得的平均OD值代表100%的寄生虫存活和生长,因此抑制率为0%。
    7. 基于表示100%增长的平均值,计算各种浓度的TPF的增长百分比以及相应的抑制百分比。
    8. 选择所有值并插入一个散点图,该点在x轴上描述TPF浓度,在y轴上描述抑制百分比。
      注意:y轴上的值表示为0到1之间的实数。
    9. 将x轴设置为对数刻度,并且最终图形必须在某个范围内近似线性。
      注意:计算IC 50 值的线性回归方法基于以下事实:计算出的抑制百分比大于0%且小于100%。该方法在整个剂量反应曲线中都假设为线性关系,这种情况很少见,因为它通常呈S型。为了将这种方法用于正确的IC 50 计算,通常必须将其与一个或两个轴的对数转换结合使用,以确保正确的IC剂量反应曲线转化为线性近似值(Nevozhay,2014年)。
    10. 选择“添加趋势线”并选择“线性”趋势线选项。
      注意:趋势线的R平方值等于或接近1时最可靠。
    11. 使用此图上的线性(y = ax + b)方程,对于y = 0.5值,x点等于IC 50 值。 IC 50 值以µg / ml表示。

  4. 选择性指数(SI)的确定
    选择性指数(SI)定义为J774A.1巨噬细胞的50%细胞毒性浓度与利什曼原虫 spp的50%抑制浓度之比。 amastigotes(CC 50 / IC 50 )。被测化合物对细胞内利什曼原虫 spp的体外活性。鼠类巨噬细胞缺乏对小鼠巨噬细胞的明显细胞毒性(SI> 1),表明其具有治疗利什曼病的潜力(Makwali et al。,2015)。

笔记

建议在首次进行测定之前优化实验参数,例如孵育时间,细胞和寄生虫的数量以及刃天青的用量,因为这些参数会影响代谢测定的一致性(Czekanska,2011; Kim和Jang,2018)。而且,刃天青和试卤灵对光敏感并且需要避光保护,否则会导致灵敏度降低。

菜谱

  1. 胎牛血清(FBS)
    1. 解冻FBS,并在不断搅拌下在搅拌的水浴中于56°C加热灭活30分钟。
    2. 储存在-20°C直到使用
  2. 完整的RPMI-1640培养基
    1. 加入5 ml的L-谷氨酰胺(储备溶液200 mM,在-20°C下储存),5 ml青霉素-链霉素(储备溶液10,000 U / ml,在-20°C下储存)和5 ml HEPES(储备溶液1 M)至500 ml RPMI-1640培养基
    2. 储存在4°C
    3. 取适当的体积,并添加FBS至最终浓度的10%v / v
  3. 完整填写施耐德的媒体
    1. 加入5 ml青霉素-链霉素(储存在10,000°C / ml的-20°C的溶液中)
    2. 储存在4°C
    3. 取适当的体积,将FBS添加至最终浓度的20%v / v
  4. 磷酸盐缓冲液(PBS),10x,pH = 7.2-7.4
    1. 将800毫升蒸馏水放入合适的容器中,溶解80克NaCl,2克KCl,11.5克Na 2 HPO 4 和2克KH 2 PO 4
    2. 搅拌直至盐完全稀释,并根据需要用NaOH(1 M)或HCl(1 M)溶液将溶液调节至所需的pH(通常为7.2-7.4)
    3. 加入无菌和蒸馏水直至最终体积为1,000 ml,并通过孔径为0.22 µm的注射器过滤器单元进行灭菌
    4. 通过在450 ml无菌和蒸馏水中稀释50 ml的10x储备液来制备1x的工作溶液
    5. 储存在4°C
  5. 刃天青溶液
    1. 将所需量的刃天青钠盐溶解在所需的PBS 1x最终体积中(例如,将2.46 mg刃天青在1 ml PBS 1x中溶解以得到中间溶液,然后每孔稀释5 µl为了达到60 µg / ml的最终浓度)
    2. 在实验过程中避光
    3. 每次使用前都必须新鲜配制工作浓度的刃天青溶液(捷克,2011年)
  6. 0.4%(w / v)台盼蓝拒染染料
    一种。将0.4 g的锥虫蓝溶于100 ml的PBS 1x中。搅动
    b。通过打褶的滤纸过滤,并通过0.22 µm孔径的注射器过滤器单元进行消毒
    C。室温存放
  7. 0.01%(w / v)十二烷基硫酸钠(SDS)
    一种。在100 ml蒸馏水中稀释0.01 g SDS。搅动
    b。室温保存
  8. 裂解液
    在RPMI-1640培养基中稀释4.8 µl HEPES(1 M储备液)和6 ml 0.01%SDS溶液,以使最终体积为10 ml

致谢

这项研究工作的一部分由希腊研究与创新基金会(HFRI)和研究与技术总秘书处(GSRT)根据HFRI博士奖学金(GA。编号6ΝΔΘ46ΨZn2Ν-ΣΣΟ)的支持。这项工作还得到了KRHPIS II(MIS 5002486)和EATRIS(MIS 5028091)的支持。
 该协议是根据Koutsoni等人(2018)和Kyriazis等人(2013)进行改编和修改的。

利益争夺

作者宣称他们没有竞争利益。

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引用:Koutsoni, O. S., Karampetsou, K. and Dotsika, E. (2019). In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values. Bio-protocol 9(21): e3410. DOI: 10.21769/BioProtoc.3410.
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