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Jun 2019
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Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi
人畜共患病疟原虫——诺氏疟原虫增殖和生长抑制活性测定   

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Abstract

Malaria remains a major cause of morbidity and mortality globally. Clinical symptoms of the disease arise from the growth and multiplication of Plasmodium parasites within the blood of the host. Thus in vitro assays to determine how drug, antibody and genetic perturbations affect the growth rate of Plasmodium parasites are essential for the development of new therapeutics and improving our understanding of parasite biology. As both P. falciparum and P. knowlesi can be maintained in culture with human red blood cells, the effect of antimalarial drugs and inhibitory antibodies that target the invasion or growth capacity of Plasmodium parasites are routinely investigated by using multiplication assays or growth inhibition activity (GIA) assays against these two species. This protocol gives detailed step-by-step procedures to carry out flow cytometry-based multiplication assays and growth inhibition activity assays to test neutralizing antibodies based on the activity of the parasite enzyme lactate dehydrogenase of Plasmodium knowlesi adapted to human red blood cell culture. Whilst similar assays are well established for P. falciparum, P. knowlesi is more closely related to all other human infective species (Pacheco et al., 2018) and so can be used as a surrogate for testing drugs and vaccines for other malaria species such as P. vivax, which is the most widespread cause of malaria outside of Africa, but cannot yet be cultured under laboratory conditions.

Keywords: Plasmodium knowlesi (诺氏疟原虫), Malaria (疟疾), Multiplication assay (增殖测定), Growth inhibition activity assay (生长抑制活性测定), Drug screening (药物筛选), Invasion (侵入)

Background

Plasmodium blood stage parasites are responsible for the clinical symptoms of malaria, including high periodic fever and anemia. Plasmodium merozoites invade red blood cells, grow and multiply intracellularly until the blood cells burst and daughter merozoites are released to infect new red blood cells. The merozoite is briefly extracellular between egress and invasion of red blood cells, and this is a key target for blood stage vaccines as it is directly exposed to antibodies in the blood. Several invasion proteins are in the focus of vaccine development including Plasmodium falciparum merozoite surface protein 1 (MSP1), MSP2, apical membrane antigen 1 (AMA1), reticulocyte binding protein homologue 5 (RH5), erythrocyte binding antigen (EBA-175) and Plasmodium vivax Duffy binding protein (PvDBP) (Genton et al., 2003; Thera et al., 2011; Koram et al., 2016; Payne et al., 2017a and 2017b).

Investigation of antimalarial drugs or inhibitory antibodies that target the blood stages of Plasmodium parasites are carried out by using multiplication or growth inhibition activity (GIA) assays. Flow cytometry-based invasion/multiplication rate assays have previously been described for various Plasmodium species (Basco et al., 1995; Bhakdi et al., 2007; Xie et al., 2007; Izumiyama et al., 2009; Bei et al., 2010; Moon et al., 2013). The advantage of flow cytometry-based assays is that they provide absolute measurements of parasite numbers, so are particularly good for examining changes in parasitemia between different parasite lines. As parasitemia increase only occurs after the release of merozoites from schizonts resulting in invasion and formation of new ring stage parasites–the flow cytometry-based assays are particularly useful for examining this transition. They can be expressed either as % of control, or as an absolute value like fold-growth or parasite multiplication rate.

The activity of the enzyme lactate dehydrogenase (LDH) was first measured as a means to detect the presence of Plasmodium falciparum as a fast alternative to microscopy screening (Makler and Hinrichs, 1993). The LDH assay is based on the fact that Plasmodium LDH can rapidly convert lactate to pyruvate by employing the NAD analog 3-acetylpyridine NAD (APAD) as a coenzyme, whereas human erythrocyte LDH uses APAD instead of NAD at a much smaller rate (200-fold slower). Measuring the malarial LDH activity in the presence of APAD is a specific and sensitive method for the detection of Plasmodium parasites (Basco et al., 1995).

The standardized P. falciparum growth inhibition activity assays based on the LDH activity are used as standard to investigate vaccine candidate antigen activity (Kennedy et al., 2002) and routinely used for analysis of clinical trials with the international GIA Reference Laboratory at the NIH, USA (Malkin et al., 2005). LDH activity assays are often simpler than flow cytometry-based assays, but measure the presence of parasite derived LDH rather than directly measuring the number of infected cells. As such they can only provide relative growth rate values, so are best suited for comparison of multiple treatments of specific parasite lines e.g. for drug or antibody inhibition assays. Relative growth rates are normally expressed as a % of an untreated control.

This protocol describes the methodology for both multiplication assays based on flow cytometry and GIA assays based on LDH activity of Plasmodium blood stage parasites (Figure 1). Here, the focus is on the simian malaria parasite Plasmodium knowlesi, but they can easily be adapted to other Plasmodium species by adjusting incubation times based on the life cycle length and specific culture conditions. P. knowlesi has recently been adapted to grow in human Duffy positive blood (Moon et al., 2013) and is therefore the second human malaria parasite with a long-term in vitro culture system, next to P. falciparum. Due to its close ancestry and biology, it is a suitable model to study invasion genes of P. vivax, which lacks a long-term in vitro culture system.



Figure 1. Schematic showing the procedures of multiplication and growth inhibition assays for Plasmodium knowlesi

Materials and Reagents

  1. 1.5 ml micro centrifuge tube (Eppendorf, catalog number: 0030120086)
  2. 15 ml centrifuge tube (Falcon, Corning, catalog number: 352196)
  3. 24-well plates (CytoOne, Starlab, catalog number: CC7672-7524)
  4. 96 flat-bottom plates (CytoOne, Starlab, catalog number: CC7672-7596)
  5. 96-well flat/half area tissue culture cluster plates (Corning, catalog number: 3697)
  6. Aluminium foil
  7. Plasmodium knowlesi A1-H.1 wild type (Mike Blackman, Francis Crick Institute London) (Moon et al., 2013)
  8. Duffy positive (Fy+) human red blood cells
  9. RPMI-1640 Media (Sigma-Aldrich, catalog number: R5886)
  10. L-glutamine (Sigma, catalog number: G7513-100ML), -20 °C
  11. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761)
  12. Dextrose (Sigma, catalog number: G7021)
  13. Hypoxanthine (Sigma-Aldrich, catalog number: H9636)
  14. Albumax II (Gibco, catalog number: 11560376)
  15. Horse serum (PAN BIOTECH, catalog number: P30-0711), -20 °C
  16. Nycodenz (Progen, catalog number: 1002424), room temperature
  17. Paraformaldehyde (Pierce 16% Formaldehyde [w/v]) (Thermo Fisher, catalog number: 28906) room temperature
  18. PBS tablets (MP BiomedicalsTM, catalog number: MP2810305), room temperature
  19. Glutaraldehyde Solution Grade I, 25% in water (Sigma-Aldrich, catalog number: G5882-10X1ML)
  20. Triton X-100 (Roche, Sigma, catalog number: 11332481001)
  21. Ribonuclease A (MP BiomedicalsTM, catalog number: 0219398050)
  22. SYBR® Green I nucleic acid gel stain (Life Technologies, Sigma-Aldrich, catalog number: S9430-5ML)
  23. Tris HCl, Trizma® hydrochloride solution 1 M, pH 8.0, (Sigma-Aldrich, catalog number T3038-1L)
  24. Sodium L-lactate (Sigma-Aldrich, catalog number: 71718)
  25. 3-acetylpyridine adenine dinucleotide (APAD) (Sigma-Aldrich, catalog number: A5251)
  26. Phenazine ethosulfate (Sigma-Aldrich, catalog number: P4544)
  27. Nitro Blue Tetrazolium tablets (Sigma-Aldrich, catalog number: N5514)
  28. 4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine; compound 2 (Michael Blackman, Francis Crick Institute, London, UK)
  29. Diaphorase from Clostridium klyveri (Sigma-Aldrich, catalog number: D5540-1.5KU)
  30. Custom Modified RPMI media w/o glutamine (Life Technology Brand, see Recipes), 4 °C
  31. Fixative: 4% paraformaldehyde with 0.4% glutaraldehyde (see Recipes)
  32. LDH substrate buffer, pH 7.5 (see Recipes)
  33. Nitro Blue Tetrazolium (NBT) solution (see Recipes)
  34. 3-Acetylpyridine Adenine Dinucleotide (APAD) stock solution (10 mg/ml) (see Recipes)
  35. Diaphorase stock solution 50 units/ml (see Recipes)

Equipment

  1. Becton Dickenson LSR-II Flow Cytometer or equivalent
  2. Upright binocular compound light microscope with 100x oil objective
  3. Multichannel pipette (8-Channel Pipette, 30-300 μl) (ErgoOne®, catalog number: S7108-3300)
  4. Plate centrifuge (Eppendorf, model: 5810R, catalog number: 5811000660)
  5. Titramax 100 Flatbed shaker (Heidolph Instruments, catalog number: 544-11200-00)
  6. Microplate Spectrophotometer Spectra Max 340P
  7. Class II Microbiological Safety Cabinet
  8. -20 °C freezer
  9. -70 °C freezer

Software

  1. FACSDiva 6.1.3 software
  2. FlowJo, https://www.flowjo.com/
  3. Flowing software, http://flowingsoftware.btk.fi/

Procedure

The protocol of thawing Plasmodium knowlesi A1-H.1 parasite depends on the source of parasites and needs to be checked with the person that froze the sample. Parasites are maintained in a flask gassed with a mixture of 90% N2, 5% O2 and 5% CO2 at 37 °C, monitored by microscopy using Giemsa-stained thin films, and parasitaemia maintained at between 0.5% and 10%. All procedures need to be carried out with sterile equipment, materials and reagents with aseptic techniques. Local safety policies must be followed for all work involving human infectious agents.


  1. Parasite multiplication assay
    1. Synchronize P. knowlesi parasites via purification with Nycodenz
      1. Transfer 5 ml of 55% Nycodenz working solution to a 15 ml conical tube and warm up to room temperature (check Note 1).
      2. Centrifuge down a high parasitemia (4-10%) P. knowlesi 50 ml culture with 2% hematocrit at 900 x g for 4 min at high brake/acceleration at room temperature.
      3. Resuspend parasite pellet at 50% hematocrit in RPMI.
      4. Carefully lay 2 ml of this culture onto 5 ml Nycodenz in a 15 ml tube.
      5. Centrifuge at 900 x g for 12 min with low brake/acceleration (check Note 2).
      6. Transfer the brownish colored top layer schizonts to a new conical tube and wash with RPMI to remove Nycodenz (see Figure 2).
      7. Incubate schizonts in culture media with 1 μM Compound 2 for 2-3 h (check Note 3).
      8. Wash off Compound 2 with RPMI (centrifuge at 900 x g for 4 min at high brake/acceleration at room temperature) and transfer schizonts back to culture (with 2% hematocrit red blood cells). The number of harvested schizonts depends on the starting parasitemia and age of the parasites (check Note 4).


        Figure 2. Schizont enrichment with Nycodenz

    2. In the following cycle, when parasites are again reaching the schizont stage, set up the multiplication assay in a 96-well plate (see Figure 3 for the plate layout).
      1. Fill out all outer wells in the 96-well plate with 100 μl of RPMI or sterile water. This ensures inner wells are not affected by evaporation.
      2. Plate out 75 μl complete growth media alone (or with vehicle control) or with 2x concentration of drug or antibody to be tested. Plate out 75 µl of the parasite culture per well as indicated in the plate layout. The plate layout can be customized depending on the individual experiment (see Figure 1 for plate layout).
      3. Prepare a culture with young schizonts at around 1% parasitemia and 4% hematocrit.
        1. Purify synchronous schizonts with Nycodenz as described in Step A1.
        2. Resuspend parasite pellet with 1 pellet-volume complete media to 50% hematocrit.
        3. Prepare complete media with 4% red blood cells (e.g., 5 ml media + 200 μl red blood cells).
        4. Transfer 1% schizonts to media + 4% red blood cells (around 4-6 μl of 50% schizonts to 5 ml, depending on the parasitemia of enriched schizonts that is usually between 80 and 90%) and mix well.
        5. Confirm the parasitemia by counting at least 400 cells of a blood smear.
      4. Add 75 μl of the culture to each well that contains 75 μl media and mix by pipetting.
      5. Plate out 150 μl of uninfected red blood cells (2% hematocrit) as a control.
      6. Mix by pipetting up and down and transfer 50 μl of each well to a new 96-well plate. Carry out Step A3. if the parasites can be measured with FACS within the next 30 min or with Step A4. if parasites are fixed and measured up to 1 week later (check Notes 5, 6, 7).
      7. Incubate the 96-well plate with 100 μl parasite cultures for one growth cycle (24 h) under standard parasite culture conditions (37 °C, 3% O2 and 3% CO2 and 94% Nitrogen). 
      8. After 24 h incubation, mix each well and transfer 50 μl of the remaining 100 μl culture to a new 96-well plate. This is plate 2 (final parasitemia). Continue with Step A3. if the parasites can be measured with FACS within the next 30 min or with Step A4. if parasites are fixed and measured up to 1 week later (check Notes 5, 6, 7).


        Figure 3. Example of the plate layout

    3. Prepare samples for flow cytometry for live cell flow cytometry
      1. Prepare filtered PBS and a 1:5,000 dilution of SYBR Green I in PBS.
      2. Add 50 μl of SYBR Green I dilution to the 50 μl of parasite culture and incubate for exactly 30 min at room temperature.
      3. Dilute stained cultures 1:5 in filtered PBS by adding 40 μl of stained cells to 160 μl of PBS and run on a FACS machine.
    4. Prepare samples for fixed cell flow cytometry
      1. Prepare all buffers in filtered PBS [0.3% (v/v) Triton X-100, 0.5 mg/ml ribonuclease A, 1:10,000 dilution of SYBR Green I)].
      2. Add 50 μl of fixative to 50 μl of parasite culture and incubate at 4 °C for at least 1 h or overnight.
      3. Centrifuge down plates for 4 min, at 4 °C and 763 x g in bench top centrifuge for plates with medium brake/acceleration settings.
      4. Remove the supernatant and resuspend the cell pellet with 100 μl PBS and spin down again.
      5. Remove the supernatant and resuspend the cell pellet with 100 μl of PBS with 0.3% (v/v) Triton X-100 and incubate for 10 min at room temperature.
      6. Wash twice with 100 μl PBS.
      7. Remove the supernatant and resuspend the pellet with 100 μl 0.5 mg/ml ribonuclease A in PBS and incubate for 1 h at 37 °C.
      8. Wash with 100 μl PBS.
      9. Remove as much PBS as possible and resuspend pellet with 100 μl 1:10,000 SYBR Green in PBS and incubate for exactly 30 min at room temperature (always keep SYBR Green tube and stained cells protected from light and keep SYBR GREEN I incubation consistent for all samples).
      10. Resuspend cells again and dilute 1:5 in PBS (Transfer 40 μl to a new 96-well plate with 160 μl PBS. No need to wash off SYBR Green I).
      11. Keep plates at 4 °C and protected from light. Measure parasitemia by FACS on the same day.
    5. Record events by flow cytometry
      1. For the Becton Dickenson LSR-II the following Voltages are used:
        FSC                                               170       log
        SSC                                               209       log
        Green laser 530/30 488 F          360       log
      2. Record 50,000-100,000 cells/treatment group.
      3. Export data to fsc file format.
    6. Analyze the flow cytometry data
      1. Upload the fsc files to FlowJo, Flowing or a comparable flow cytometry software.
      2. Open Dot Plots for each well of the 96-well plate.
      3. Within the Dot Plot of the RBC only control, generate a gate for RBCs (R1) that excludes other blood cells or broken red cells. X-axis is FSC-A and y-axis is SSC-A (see Figure 2).
      4. Open a Histogram for R1 and generate a gate for parasites (H-2). X-axis is SYBR-Green signal 530/30 488F-A and y-axis is number of cells. Compare RBC only control with a parasite containing well to find the right areas on the x-axis where RBCs and parasites need to be separated (Figure 4). Use the same gates for all wells.
      5. Save data as xls file and open it with excel.
      6. Calculate ratios (H-2/R1*100) and subtract the ratio of RBC only control. Calculate the ratio of End (24 h incubation, plate 2) and Start (0 h incubation, plate 1) to determine the multiplication rate (Example in Figure 5).


        Figure 4. Gating of flow cytometry data. Within the Dot Plot a region (R1, green) defines red blood cells. All cells within that region are analyzed within a Histogram. In the Histogram a region (H-2) is defined to separate parasites from uninfected red blood cells (RBCs). The ratio of Parasite/total RBCs ((H-2/R1)*100) is calculated to give the parasitemia.


        Figure 5. Calculation of Multiplication rates

  2. Parasite growth inhibition activity (GIA) assay
    1. Set up GIA assay in 96-well plate flat/half area tissue cluster culture plates
      1. Plates should include: 
      2. Triplicate wells of each test IgG concentration
        Triplicate wells of positive control
        6 x wells with infected erythrocytes and media only (0% GIA control)
        6 x wells with uninfected erythrocytes at 2% hematocrit or iRBCs + 5 mM EDTA (100% GIA control / background)
        3 x wells with non-malarial human antibody as a negative control
      3. Plate out 20 μl of purified IgG (check Note 8) in 2x concentration in incomplete media to wells. Use serial 1:2 dilutions of immune serum or pre-immune serum of around eight final concentrations, depending on the EC50 value (for example 10, 5, 2.5, 1.25, 0.625, 0.312, 0.15 and 0.075 mg/ml). Set up each treatment in triplicate test wells.
      4.  Enrich P. knowlesi parasites via Nycodenz purification (see Step A1) or by magnetic separation (Ribaut et al., 2008).
      5. Prepare 20 ml cell culture at 1.5 % parasitemia in 4% red blood cell suspension in 2 x warm complete media (e.g., 20 ml media + 800 μl red blood cells + 12 μl trophozoites (~1.2 x 109).
      6. Confirm parasitemia by counting of blood smear. 
      7. Add 20 μl of culture with 4% hematocrit and 1.5% parasitemia trophozoites or only uninfected RBCs to the IgG plates using a multichannel pipette. Change tips for each set of triplicate samples. 
      8. Incubate for one cycle (in P. knowlesi 26-27 h) in standard culture conditions, together with a tracking culture (5 ml cell culture + 5 ml media in culture flask) to check parasitemia and life cycle stage.
      9. Before harvest check parasitemia (~ 4%) and life cycle stage (trophozoites are ideal, young parasites don’t have adequate LDH levels) of tracking culture.
    2. GIA assay harvest
      1. Add 100 μl of cold PBS to each well. 
      2. Spin down plates at 1,300 x g for 4 min with maximum acceleration/break setting. 
      3. Aspirate 100 μl of supernatant from all wells. Do not aspirate any RBCs. Tilting the plate away from the RBC pellet will help.
      4. Repeat the Steps a to conce. 
      5. Freeze at -80 °C if the assay is not going to be performed immediately–but this is not necessary otherwise.
    3. Measure parasitemia with the lactate dehydrogenase (LDH) activity assay
      1. Have NBT solution in LDH buffer ready and warm to room temperature. 
      2. If plates have been frozen, thaw them at room temperature for at least 30 min. Plates need to be uniformly warmed to room temperature. If the plates have not been frozen, it is important that RBCs are uniformly resuspended prior to assay e.g. shake plate on Titramax at max settings for 1 min and check if RBC pellets have fully disappeared (check Note 9).
      3. Prepare complete LDH substrate by adding 50 μl of 3-Acetylpyridine Adenine Dinucleotide (APAD) stock and 200 μl of Diaphorase stock to every 10 ml of NBT solution. After all reagents are combined, use prepared LDH substrate immediately. 
      4. Start the timer and add 120 μl of complete LDH substrate to all wells. Avoid bubbles (fully depress the plunger of the multichannel pipette before picking up the first aliquot of substrate, pipette should draw up extra substrate and no air will be ejected. Pipette down at the edge of the wells).
      5. Incubate plate covered with aluminium foil to protect from light and measure absorbance at 650 nm with a 96-well Microplate Spectrophotometer every 5 to 10 min until OD reaches 0.4-0.6 in iRBC and ICM control (0% GIA).
      6. Calculate percent inhibition: 100 – [(A650 of immune sample – A650 of RBCs only)/(A650 of pre-immune control – A650 of RBCs only) x 100].

Notes

  1. 5 ml Nycodenz tube is required to purify parasites from 1 ml packed red blood cells at 4-10% parasitemia (e.g., 50 ml culture maintained at 2% hematocrit and 4-10% parasitemia will yield 1 ml of packed red blood cells after centrifugation). Add 1 ml of medium to 1 ml of packed red blood cells to make a 2 ml culture with 50% hematocrit.
  2. Uninfected red cells and ring-stage parasites will sink to the bottom and schizonts form a layer on top of the Nycodenz.
  3. Compound 2 is a PKG inhibitor that reversibly blocks merozoite egress. This step is optional but will help to maximize yield of late schizonts and also provides the user with some flexibility in timing for subsequent steps. Viability of parasites will dramatically decline for incubations longer than 3 h. As an alternative to Compound 2 a highly specific and potent derivative of Compound 2, referred to as ML10, can be used as well, which is available from LifeArc (Ressurreição et al., 2020).
  4. To get parasites even more synchronized let them invade red blood cells for 30 to 60 min and purify with Nycodenz again, only this time keep the ring stage parasite pellet and remove the schizonts. You can slow down the aging of the parasites by leaving them at room temperature for several h in order to get them to the schizont stage at a convenient time for the next purification.
  5. Set up all experiments in three biological replicates (different days, parasite preparations, and RBCs).
    1. For parasite multiplication assays, setting up with purified schizonts is critical, as this removes residual uninfected RBCs from the parasites and enables us to examine differences in multiplication in different host RBCs (e.g., comparing human and macaque RBC invasion).
    2. Whilst the initial starting parasitemia should be fixed for all samples, it is still critical to obtain a timepoint 0 for parasitemia (i.e., plate 1 for multiplication assay). This is because small variations in starting parasitemia can have a significant effect on the calculated growth rate–using different RBCs between samples can also alter the precise starting parasitemia.
      Whilst here we use a second time point of 24 h, various other timepoints could be used depending on experimental aims. A timepoint of 24 h works well because all parasites would have undergone reinvasion and progressed to schizonts again, which are very easily identified using the flow cytometry assay. The downside of this is that both invasion efficiency and viability of developing rings and trophozoites are measured. Shorter timepoints (e.g., 2-6 h) can be used to more specifically look at invasion and early ring formation, but these require very synchronous and late stage schizonts, as any schizonts that have not yet progressed to form new rings at the specified timepoint will affect interpretation of the data.
  6. Parasites that are fixed become aggregated and settle more quickly to the bottom of the plate, otherwise there are no noticeable effects between fixed and unfixed samples.
  7. Assays can also be set up in 24-well plates with final volume of 1 ml.
  8. For polyclonal antibodies; purified IgG from serum is typically used and no background inhibition is usually seen from negative control samples (N.B. mouse IgG can sometimes be problematic in P. falciparum GIA assays; but all other species tested have been OK), purified IgG from plasma cannot typically be used as the anticoagulant (EDTA or heparin) cannot be fully removed even with IgG purification, leading to background GIA. P. falciparum GIA assays using diluted serum are reported, but are less common. Monoclonal antibodies work well when purified.
  9. If necessary, use a pipette to resuspend, but this poses the risk of introducing bubbles. Do not vortex).

Recipes

  1. Fixative: 4% paraformaldehyde with 0.4% glutaraldehyde
    10x PBS                                                                5 ml
    16% Formaldehyde (w/v) from Pierce                 12.5 ml
    Glutaraldehyde                                                    125 μl
    H2O                                                                     32.5 ml
    Final volume                                                        50 ml
  2. LDH substrate buffer, pH 7.5
    1. To prepare 500 ml of the buffer, mix 50 ml of 1 M Tris HCI (pH 8.0) and 450 ml H2O
    2. Add 2.8 g sodium L-Lactate
    3. Add 1.25 ml Triton X-100
    4. Mix on a magnetic stirrer at room temperature for at least 30 min
    5. Make 50 ml aliquots and freeze at -20 °C
  3. Nitro Blue Tetrazolium (NBT) solution
    1. Remove one 50 ml aliquot of LDH buffer from the freezer
    2. Warm up to room temperature.
    3. In a 50 ml tube, add 50 ml of LDH buffer to one NBT tablet (10 mg)
    4. Cover the tube with aluminium foil and  leave in fridge overnight to dissolve
    5. Do not shake! Mix gently
    6. Prepared solution can be kept at 4 °C up to 3 weeks in the dark (covered with aluminium foil)
  4. 3-Acetylpyridine Adenine Dinucleotide (APAD) stock solution (10 mg/ml)
    1. To prepare 10 ml of APAD stock solution, dissolve 100 mg of APAD in 10 ml of distilled water
    2. Store the APAD stock solution in 50 μl aliquots in PCR tubes at -20 °C
  5. Diaphorase stock solution 50 units/ml
    To prepare Diaphorase stock solution, dissolve the 1,500 units Diaphorase vial contents in 30 ml of distilled water. Store 200 μl aliquots at -20 °C
  6. Complete media
    RPMI-1640 (HEPES Modification, 25 mM HEPES, without L-glutamine, Merck) with the following additions:
    2.3 g/L sodium bicarbonate (Sigma)
    2 g/L dextrose (Sigma)
    0.05 g/L Hypoxanthine (Sigma)
    5 g/L Albumax II (Gibco)
    0.3 g/L L-glutamine (10 ml of 200 mM solution pro 1 L media, Merck)
    10% (vol/vol) horse serum
    Sterile filter and store at 4 °C

Acknowledgments

This work is supported by an MRC Career Development Award (MR/M021157/1) jointly funded by the UK Medical Research Council and Department for International Development (R.W.M, F.M), (Basco et al., 1995; Mohring et al., 2019). This work was supported in-part by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement for MultiViVax [number 733073]. T.A.R. held a Wellcome Trust Research Training Fellowship [108734/Z/15/Z]; and S.J.D. is a Jenner Investigator; a Lister Institute Research Prize Fellow and a Wellcome Trust Senior Fellow [106917/Z/15/Z]. We thank Amelia Lias and Don Van Schalkwyk for their advice on the manuscript.

Competing interests

The authors declare no competing financial interests.

Ethics

The project, consent and protocol were approved by the LSHTM Observational Research Ethics Committee under project reference 5520-1.

References

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  7. Koram, K. A., Adu, B., Ocran, J., Karikari, Y. S., Adu-Amankwah, S., Ntiri, M., Abuaku, B., Dodoo, D., Gyan, B., Kronmann, K. C. and Nkrumah, F. (2016). Safety and immunogenicity of EBA-175 RII-NG malaria vaccine administered intramuscularly in semi-immune adults: A Phase 1, double-blinded placebo controlled dosage escalation study. PLoS One 11(9): e0163066.
  8. Makler, M. T. and Hinrichs, D. J. (1993). Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am J Trop Med Hyg 48(2): 205-210.
  9. Malkin, E. M, Diemert, D. J. McArthur, J. H., Perreault, J. R., Miles, A. P., Giersing, B. K., Mullen, G. E., Orcutt, A., Muratova, O., Awkal, M., Zhou, H., Wang, J., Stowers, A., Long, C.A., Mahanty S., Miller, L.H., Saul, A. and Durbin, A.P. (2005). Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria. Infect Immun 73(6):3677-85.
  10. Mohring, F., Hart, M. N., Rawlinson, T. A., Henrici, R., Charleston, J. A., Diez Benavente, E., Patel, A., Hall, J., Almond, N., Campino, S., Clark, T. G., Sutherland, C. J., Baker, D. A., Draper, S. J. and Moon, R. W. (2019). Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife 8: 45829.
  11. Moon, R. W., Hall, J., Rangkuti, F., Ho, Y. S., Almond, N., Mitchell, G. H., Pain, A., Holder, A. A. and Blackman, M. J. (2013). Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A 110(2): 531-536.
  12. Pacheco, M. A., Matta, N. E., Valkiunas, G., Parker, P. G., Mello, B., Stanley, C. E., Lentino, M., Garcia-Amado, M. A., Cranfield, M., Kosakovsky Pond, S. L. and Escalante, A. A. (2018). Mode and rate of evolution of haemosporidian mitochondrial genomes: timing the radiation of avian parasites. Mol Biol Evol 35(2):383-403. 
  13. Payne, R. O., Silk, S. E., Elias, S. C., Miura, K., Diouf, A., Alanine, D., Jin, J., Labbe, G., Brian, I., Poulton, I., Griffiths, O., Edwards, N., Berrie, E., Siani, L., Douglas, A., Roberts, R., Vekemans, J., Nugent, F., Hill, A. V., Long, C., Lawrie, A. M. and Draper, S. J. (2017a). Safety and immunogenicity of the novel Plasmodium falciparum blood-stage vaccine chad63-mva rh5 in a phase ia clinical trial. Am J Trop Med Hyg 95(5): 313-313.
  14. Payne, R. O., Silk, S. E., Elias, S. C., Milne, K. H., Rawlinson, T. A., Llewellyn, D., Shakri, A. R., Jin, J., Labbe, G. M., Edwards, N. J., Poulton, I. D., Roberts, R., Farid, R., Jorgensen, T., Alanine, D. G., de Cassan, S. C., Higgins, M. K., Otto, T. D., McCarthy, J. S., de Jongh, W. A., Nicosia, A., Moyle, S., Hill, A. V., Berrie, E., Chitnis, C. E., Lawrie, A. M. and Draper, S. J. (2017b). Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies. JCI Insight 2(12): e93683.
  15. Ressurreição, M., Thomas J.A., Nofal S.D., Flueck, C., Moon, R.W., Baker, D.A., van Ooij, C. (2020). Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization of Plasmodium falciparum and Plasmodium knowlesi asexual stage parasites. bioRxiv. doi.org/10.1101/2020.04.24.059493.
  16. Ribaut, C., Berry, A., Chevalley, S., Reybier, K., Morlais, I., Parzy, D., Nepveu, F., Benoit-Vical, F. and Valentin, A. (2008). Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J 7: 45.
  17. Thera, M. A., Doumbo, O. K., Coulibaly, D., Laurens, M. B., Ouattara, A., Kone, A. K., Guindo, A. B., Traore, K., Traore, I., Kouriba, B., Diallo, D. A., Diarra, I., Daou, M., Dolo, A., Tolo, Y., Sissoko, M. S., Niangaly, A., Sissoko, M., Takala-Harrison, S., Lyke, K. E., Wu, Y., Blackwelder, W. C., Godeaux, O., Vekemans, J., Dubois, M. C., Ballou, W. R., Cohen, J., Thompson, D., Dube, T., Soisson, L., Diggs, C. L., House, B., Lanar, D. E., Dutta, S., Heppner, D. G., Jr. and Plowe, C. V. (2011). A field trial to assess a blood-stage malaria vaccine. N Engl J Med 365(11): 1004-1013.
  18. Xie, L., Li, Q., Johnson, J., Zhang, J., Milhous, W. and Kyle, D. (2007). Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria. Parasitology 134(Pt 9): 1151-1162.

简介

[摘要] 疟疾仍然是全球发病率和死亡率的主要原因。该疾病的临床症状源于宿主血液中疟原虫的生长和繁殖。因此,体外测定以确定药物,抗体和遗传扰动如何影响疟原虫寄生虫的生长速率对于开发新疗法和增进我们对寄生虫生物学的理解至关重要。由于两个恶性疟原虫和P. knowlesi 可以在培养物中维持与人体红细胞,抗疟疾药物和抑制性抗体靶向的侵袭能力的影响疟原虫寄生虫 是通过使用针对这两个物种乘法测定或生长抑制测定法常规地研究。该协议给出了详细的一步一步的过程来进行基于所述寄生虫乳酸脱氢酶的活性为基础的流式细胞仪乘法测定和生长抑制活性测定法测试性中和抗体的疟原虫knowlesi 适于人类红血细胞培养物中。虽然类似测定法是用于很好地建立的恶性疟原虫,P. knowlesi 被更密切相关的所有其他人类感染性物种(帕切科等人。,2018),因此可以用作替代用于测试药物和疫苗用于其它疟疾种类,例如如间日疟原虫,它是非洲以外疟疾最广泛的病因,但尚未在实验室条件下进行培养。

[背景 ] 疟原虫血阶段寄生虫负责疟疾的临床症状,包括高周期性发热和贫血。疟原虫裂殖子侵入红细胞,在细胞内生长并繁殖,直到血细胞破裂并释放子裂殖子以感染新的红细胞。裂殖子在红细胞的流出和侵入之间短暂地在细胞外,这是血液阶段疫苗的关键靶标,因为它直接暴露于血液中的抗体。几个侵入蛋白是疫苗开发的包括焦点恶性疟原虫裂殖子表面蛋白1 (MSP1) ,MSP2,顶膜抗原1,网织红细胞结合同源物5,红细胞结合抗原(EBA-175)和聚乳酸smodium间日疟原虫达菲结合蛋白(Genton先生等人,2003;锡拉等人,2011;韩美。等人,2016;佩恩等人,2017年和2017年b )。

通过使用增殖或生长抑制活性测定法来 研究针对疟原虫疟原虫血液阶段的抗疟药或抑制性抗体。流基于流式细胞术入侵/倍增率的测定之前已经为各种所描述的疟原虫物种(巴斯科等人,1995; Bhakdi 等人,2007;解等人,2007;泉山。等人,2009;贝等。,2010; Moon 等人,2013)。基于流式细胞仪的测定法的优势在于它们提供了寄生虫数量的绝对测量值,因此特别适合检查不同寄生虫系之间的寄生虫血症变化。由于寄生虫血症的增加仅发生在裂殖子裂殖子释放导致新的环形阶段寄生虫入侵和形成之后,因此基于流式细胞仪的测定对于检查这种转变特别有用。它们可以表示为对照的百分比,也可以表示为绝对值,如倍数增长或寄生虫繁殖率。

乳酸脱氢酶(LDH)的活性首先被测量为检测恶性疟原虫的存在的手段,作为显微镜筛选的快速替代方法(Makler和Hinrichs,1993)。LDH分析基于以下事实:疟原虫LDH可通过使用NAD类似物3-乙酰基吡啶NAD(APAD)作为辅酶将乳酸迅速转化为丙酮酸,而人红细胞LDH使用APAD而不是NAD的速率要小得多(200-折叠较慢)。在APAD存在下测量疟疾LDH活性是检测疟原虫寄生虫的一种特殊而灵敏的方法(Basco 等,1995)。

基于所述LDH活性的生长抑制活性的测定法被用作标准来调查疫苗候选抗原活性(肯尼迪等人升。,2002) 。LDH活性测定通常比基于流式细胞术的测定更简单,但是要测量寄生虫衍生的LDH的存在,而不是直接测量被感染细胞的数量。因此,它们只能提供相对生长速率值,因此最适合比较特定寄生​​虫系的多种治疗方法,例如用于药物或抗体抑制试验。相对增长率通常表示为未经处理的对照组的百分比。

该协议描述了基于流式细胞术的增殖测定和基于疟原虫血阶段寄生虫的LDH活性的生长抑制测定的方法学(图1)。在此,焦点位于猿猴疟疾PA rasite 疟原虫knowlesi ,但他们可以很容易地适应其它疟原虫物种通过基于生命周期的长度和特定的培养条件调节温育时间。P. knowlesi 最近被dapted人类达菲阳性血液生长(月亮等人,2013),因此是第二人疟疾寄生虫具有长期体外培养体系,毗邻的恶性疟原虫。由于它的近缘和生物学特性,它是研究间日疟原虫入侵基因的合适模型,间日疟原虫缺乏长期的体外培养系统。



D:\ Reformatting \ 2020-7-1 \ 1902764--1483 Robert Moon 837224 \ Figs jpg \ Figure1.jpg

图1 。示意图显示诺氏疟原虫的增殖和生长抑制测定方法

关键字:诺氏疟原虫, 疟疾, 增殖测定, 生长抑制活性测定, 药物筛选, 侵入

材料和试剂


 


1.5 ml 微量离心管(Eppendorf,目录号:0030120086)
15 ml离心管(Falcon,康宁,目录号:352196)
24 - 孔板(CytoOne ,STARLAB ,目录号:CC7672-7524)
96个平底板(CytoOne ,Starlab ,目录号:CC7672-7596)
96 - 孔平/半区域的组织培养群集板(Corning,目录号:3697)
铝合金箔
疟原虫knowlesi A1-H.1 野生型(迈克·布莱克曼,弗朗西斯·克里克研究所伦敦)(月亮等人。,2013)
达菲阳性(Fy +)人类红细胞
RPMI -1640介质(Sigma-Aldrich,目录号:R5886)
L-谷氨酰胺(Sigma,目录号:G7513-100ML),- 20 °C
碳酸氢钠(Sigma -Aldrich ,目录号:S5761)
葡萄糖(Sigma,目录号:G7021)
次黄嘌呤(Sigma -Aldrich ,目录号:H9636)
Albumax II(Gibco,目录号:115603 76)
马血清(PAN BIOTECH,目录号P30-0711),-20 °C
Nycodenz (Progen ,目录号:1002424),室温
室温下的多聚甲醛(皮尔斯16%甲醛[ w / v ] )(Thermo Fisher,目录号:28906)
PBS片剂(MP 生物体检TM ,目录号:MP2810305),室温下
I 级Glutara 乙醛溶液,在水中25%(Sigma -Aldrich ,目录号:G5882-10X1ML)
Triton X-100(Roche,Sigma,目录号:11332481001)
核糖核酸酶A(MP Biomedicals TM ,目录号:0219398050)
SYBR ® 绿色我核酸凝胶染色(Life Technologies公司,Sigma-Aldrich公司,目录号:S9430-5ML)
三HC 升,氨基丁三醇® 盐酸盐溶液的1M ,pH 8.0中,(Sigma-Aldrich公司,目录号T3038-1L)
L-乳酸钠(Sigma-Aldrich,目录号:71718)
3-乙酰基吡啶腺嘌呤二核苷酸(APAD)(Sigma-Aldrich,目录号:A5251)
吩嗪乙硫酸盐(西格玛奥德里奇,目录号:P4544)
硝基四唑鎓片(Sigma-Aldrich,目录号N5514)
4- [7-[(二甲基氨基)甲基] -2-(4-氟苯基)咪唑并[1,2-a]吡啶-3-基]嘧啶-2-胺;大院2(Michael Blackman,英国伦敦弗朗西斯·克里克学院(Francis Crick Institute)
心肌黄酶从梭菌klyveri (Sigma-Aldrich公司,CATAL OG号:D5540-1.5KU)
不含谷氨酰胺的定制改性RPMI介质(生命技术品牌,请参见食谱),4 °C
固定剂:4%低聚甲醛和0.4%戊二醛(请参阅食谱)
LDH底物缓冲液,pH 7.5(请参见配方)
硝基蓝四唑(NBT)解决方案(请参阅食谱)
3-乙酰基吡啶腺嘌呤D 核苷酸(APAD)储备溶液(10 mg / ml)(请参阅食谱)
心肌黄酶储备溶液50单位/毫升(请参阅食谱)
 


设备


 


Becton Dickenson LSR-II 流式细胞仪或同等功能
具有100x油镜的立式双目复合光学显微镜
多道移液器(8通道移液器,30-300 μ 升)(ErgoOne ® ,目录号:S7108-3300)
平板离心机(Eppendorf ,型号:5810R,货号:5811000660)
Titramax 100平板振动筛(Heidolph Instruments,目录号:544-11200-00 )
微孔板分光光度计Spectra Max 340P
二级微生物安全柜
-20 °C冷冻室
-7 0 °C冷冻室
 


软件


 


FACSDiva 6.1.3软件
FlowJo ,https: //www.flowjo.com/
流动软件,http://flowingsoftware.btk.fi/
程序


 


解冻的协议疟原虫knowlesi A1-H.1寄生虫取决于寄生虫和需要的源极到与该冻结了样品的人来检查上。寄生虫维持在具有90%N的混合物除气的烧瓶中2 ,5%氧气2 和5%CO 2 在37 ℃下,监测通过使用吉姆萨染色的薄膜显微镜和寄生虫血症保持在0.5和10%之间。所有程序都需要使用无菌设备,材料和试剂以及无菌技术进行。所有涉及人类传染源的工作都必须遵守当地的安全政策。


 


寄生虫增殖测定
同步P. knowlesi 经由与纯化寄生虫Nycodenz
将5 ml的55%Nycodenz 工作溶液转移到15 ml的锥形管中,并加热到室温(注意1)。
离心机向下高寄生虫血症(4-10%)P. knowlesi 50ml培养用2%的血细胞比容,在900 ×g下在4分钟高制动/加速在室温下。
在RPMI中以50%的血细胞比容重悬寄生物沉淀。
小心放置2ml该文化性的ë到5毫升Nycodenz 在一个15毫升管。
在低制动/加速度下以900 xg离心12分钟(请注意注2)。
转移带褐色的共lored顶层裂殖到一个新的锥形管中并用RPMI洗涤,REM OVE Nycodenz (参见图2)。
将裂殖体与1μM 化合物2 在培养基中孵育2-3小时(请注意注释3)。
用RPMI (在室温高制动/加速下于900 xg离心4分钟)冲洗化合物2,然后将schi zonts 转移回培养物中(含2%的红细胞比容红细胞)。收获的裂殖体的数量取决于开始的寄生虫病和寄生虫的年龄(请参阅注4)。
 


D:\ Reformatting \ 2020-7-1 \ 1902764--1483 Robert Moon 837224 \ Figs jpg \ Figure2.jpg


图2 。SC hizont富集Nycodenz


 


在下面的CYCL E,当寄生虫再次到达裂殖体阶段,设置在乘法测定96 - 孔平板(参见图3的板布局)。
在96填写所有外井- 与100孔板微升RPMI或无菌水。这确保了内部井不受蒸发的影响。
75积垢微升单独的完全生长培养基(或载体对照)或用2x浓度的药物或抗体进行测试。如板布局所示,每孔接种75 µl寄生虫培养物。可以根据各个实验定制板布局(板布局见图1)。
用大约1 %的寄生虫血症和4%的血细胞比容准备带有年轻裂殖体的培养物。
如步骤A1 所述,用Nycodenz纯化同步片段。
用1个沉淀体积的完全培养基重悬寄生虫沉淀至50%的血细胞比容。
准备含有4%红细胞的完整培养基(例如5 ml培养基+ 200μl 红细胞)。
将1 %的schizonts 转移至培养基+ 4%的红细胞中(大约4-6 µl 的50%schizonts转移至5 ml ,这取决于浓缩的schizonts的寄生虫病,通常介于80%到90%之间)并充分混合。
通过计数至少400个细胞涂片检查来确认寄生虫病。
加入75 微升的文化为包含75个每孔微升媒体和混合用移液。
镀出150 微升未感染红血细胞(2%血细胞比容)作为对照的。
移液器向上和Dow混合n和转移50 微升的每个孔中,以一个新的96 - 孔平板。执行步骤A3。是否可以在接下来的30分钟内通过FACS或步骤A4测量寄生虫。如果寄生虫已固定并在1周后进行了测量(请参见注释5、6、7)。
孵育96 - 与孔板100 微升寄生虫培养物用于一个生长周期(24小时)标准寄生虫培养条件下(37 ℃,3%氧气2 和3%CO 2 和94%氮气)。
孵育24小时后,以及混合每50传送微升剩余100的μ 升培养到一个新的96 - 孔平板中。这是板2(最终寄生虫血症)。与继续小号TEP 甲3.如果寄生虫可以用FACS在接下来的30分钟内或与被测量小号TEP 甲4.如果p arasites是固定的,后测量长达1周(检查注释5,6,7 )。
 


D:\ Reformatting \ 2020-7-1 \ 1902764--1483 Robert Moon 837224 \ Figs jpg \ Figure3.jpg


图3.板布局示例


 


准备用于流式细胞术的样品进行活细胞流式细胞术
准备过滤的PBS和PBS中SYBR Green I的1:5,000稀释液。
加入50 微升的SYBR Green稀释我的于50 微升寄生虫培养和孵化的在室温下恰好30分钟。
通过将40μl 染色的细胞添加到160μlPBS中,在过滤的PBS中稀释染色的培养物1:5,并在FACS机器上运行。
准备用于固定细胞流式细胞术的样品
在过滤的PBS中准备所有缓冲液[ 0.3%(v / v)Triton X-100,0.5 mg / ml核糖核酸酶A,SYBR Green I的1:10,000稀释液 ] 。
将50μl 的固定剂添加到50μl 的寄生虫培养物中,并在4 °C 孵育至少1 h或过夜。
在4 °C和763 xg 的台式离心机中对具有中等制动/加速设置的板将板向下离心4分钟。
除去上清液,并用100重悬细胞沉淀微升PBS中,并再次离心。
除去上清液,并用100μl 含0.3%(v / v)Triton X-100的PBS重悬细胞沉淀,并在室温下孵育10分钟。
用100洗两次微升PBS。
去除上清,重悬沉淀与100 μ 升 0.5毫克/毫升核糖核酸酶A在PBS中孵育在37 1个小时℃。
用100μlPBS洗涤。
除去尽可能多的PBS,然后用100μl1 :10,000 SYBR Green重悬沉淀,并在室温下温育30分钟(始终保持SYBR Green管和染色细胞避光,并保持SYBR GREEN I的温育始终如一样本)。
再次将细胞重悬并稀释1:PBS中的5(转印40 μ l至新的96 - 160孔板μ 。升PBS 无需洗掉的SYBR Green I)。
将板保持在4 °C 并避光。在同一天通过FACS测量寄生虫病。
通过流式细胞仪记录排放
对于Becton Dickenson LSR-II,使用以下电压:
FSC 170 日志                                         


SSC 209 日志                           


绿色激光530/30 488 F 360 日志                           


记录50,000-100,000个细胞/处理组。
将数据导出为fsc 文件格式。
分析流式细胞仪数据
将fsc 文件上传到FlowJo ,Flowing或类似的流式细胞仪软件。
打开点图对于每个孔96的- 孔板中。
在仅RBC的点图内,为RBC(R1)生成一个门,该门排除其他血细胞或破碎的红细胞。X轴为FSC-A,y轴为SSC-A(见图2)。
打开R1的直方图并生成寄生虫(H-2)的门。X轴是SYBR-Green信号530/30 488F-A,y轴是单元数。将纯红细胞对照与含有寄生虫的孔进行比较,以在x轴上找到需要分离红细胞和寄生虫的正确区域(图4)。对所有孔使用相同的浇口。
将数据另存为xls 文件,然后使用excel将其打开。
计算比率(H-2 / R1 * 100),然后减去仅RBC控制的比率。计算End(24 h孵育,板2 )和Start(0 h孵育,板1 )的比率,以确定繁殖率(图5中的示例)。
 


D:\ Reformatting \ 2020-7-1 \ 1902764--1483 Robert Moon 837224 \ Figs jpg \ Figure4--word.jpg


图4.流式细胞仪数据门控。在点图中,区域(R1,绿色)定义了红细胞。在直方图中分析该区域内的所有单元格。在直方图中,将区域(H-2)定义为将寄生虫与未感染的红细胞(RBC)分开。计算寄生虫/总红细胞的比例((H-2 / R 1)* 100)以得出寄生虫血症。


D:\ Reformatting \ 2020-7-1 \ 1902764--1483 Robert Moon 837224 \ Figs jpg \ Figure5--word.jpg


图5.乘法率的计算


 


寄生虫生长抑制活性(GIA)分析
同步P. knowlesi 的寄生虫经由Nycodenz 纯化(见步骤A1)或通过磁分离(Ribaut 等人,2008) 。
设立GIA检测在96 - 孔板
板块应包括:
每个测试IgG浓度一式三份


阳性对照一式三份


仅含感染的红细胞和培养基的6个孔(0%GIA对照)


6孔未感染的红细胞,血细胞比容为2%(100%GIA对照/背景)


以非疟疾人抗体作为阴性对照的3个孔


镀出20 微升培养物用4%的血细胞比容和1.5%的寄生虫血症滋养体或只未感染RBC诠释ö96 - 孔平/半区域的组织培养群集板。
                            用Nycodenz 或磁铁纯化同步滋养体。
用1个沉淀体积的完全培养基重悬寄生虫沉淀至50%的血细胞比容。
在完全培养基(例如5 ml培养基+ 200μl 红细胞)中准备4%的红细胞悬液。
将纯化的裂殖体转移至含有4%红细胞的培养基(6μl 的50%裂殖体至5 ml)中,获得1.5%的寄生虫血症并充分混合。6 μ 升的50%裂殖对应于〜3×10个8 细胞。
通过计数血液涂片确认寄生虫血症。
加入20 微升的完全培养基中纯化的IgG的在2x浓度的孔中。根据EC 50 值(例如10、5、2.5、1.25、0.625、0.312、0.15和0.075 mg / mL),使用大约8种最终浓度的连续1:2稀释的免疫血清或免疫前血清。在一式三份的测试孔中设置每种处理。
在标准培养条件下与跟踪培养一起温育一个周期(在P. Knowlesi 26-27 h中),并检查跟踪寄生虫和生命周期阶段。
收获前检查追踪培养的寄生虫血症(〜4%)和生命周期阶段(滋养体是理想的,幼虫没有足够的LDH水平)。
收获细胞。
通过向每个孔中吸取100μl 冷PBS 重悬每个培养物。
以1300 xg 的速度旋转培养物4分钟,并设置中等加速/破坏速度。
从所有孔中吸取100μl 上清液。不要抽吸任何RBC。将板从RBC沉淀物上倾斜会有所帮助。
重复小号TEPS BFI 到BFIII 一次。
如果不立即进行测定,请在-7 0 °C 冷冻- 否则就不需要这样做。
用乳酸脱氢酶(LDH)活性测定法测量寄生虫病
准备LDH缓冲液中的NBT溶液,并加热至室温。
如果板已经冷冻,则在室温下解冻至少30分钟。板需要均匀加热至室温。如果未冷冻培养板,则在测定前将RBC均匀悬浮是很重要的。
通过加入50准备完整的LDH底微升的3-乙酰基吡啶腺嘌呤二核苷酸(APAD)库存和200 微升黄递酶股票每10毫升NBT溶液。合并所有试剂后,请立即使用准备好的LDH底物。
启动定时器,并添加120 微升完全LDH基板的所有孔中。避免气泡(在拾取第一等分试样的底物之前完全压下多通道移液器的柱塞,移液器应吸出多余的底物,并且不会喷射出空气。移液器在孔口向下)并完全重悬RBC(摇板)在Titramax 在1分钟,并检查RBC丸粒完全消失)(最大值设置ç 赫克注8) 。
在黑暗中孵育,以使颜色在室温下在平板振荡器上显影30至60分钟。
测量在650nm处的吸光度与96 - 孔微孔板分光光度计直到OD达到0.4 - 0.6 体-iRBC 和ICM控制(0%GIA)。
计算抑制百分比:100 - [(A 650 免疫样品的-阿650 仅RBC的)/(甲650 预免疫控制-甲650 仅红细胞)×100]。
 


笔记


 


5 米升NYCO denz 管需要为1μm纯化寄生虫升堆积的红血细胞在4-10%寄生虫血症(ê .G。 ,50米升培养维持在2%的血细胞比容和4-10%寄生虫血症将产生1米离心后收集的1 个包装的红血细胞)。将1 ml的培养基添加到1 ml的包装红细胞中,以使2 ml的培养物中含有50%的血细胞比容。
未感染的红细胞和环形阶段的寄生虫将沉入底部,裂殖体在Nycodenz的顶部形成一层。
化合物2是PKG抑制剂,可逆地阻断裂殖子的流出。该步骤是可选的,但将有助于最大程度提高后期裂殖子的产量,还为用户在后续步骤的时间安排上提供了一定的灵活性。如果孵育时间超过3小时,寄生虫的生存力将急剧下降。作为替代化合物2 化合物2高度特异性和有效的衍生物,称为ML10,也可被使用,其可购自LifeArc (Ressurrei 曹等人,2020)。
为了使寄生虫更加同步,让它们侵入红细胞30至60分钟,并再次用Nycodenz进行纯化,只有这次保持环形阶段的寄生虫沉淀并去除荆芥。您可以通过将寄生虫在室温下放置数小时来减慢寄生虫的衰老,以便在方便的时间使其进入裂殖体阶段以进行下一次纯化。
将所有实验设置为三个生物学重复(不同天数,寄生虫制备和RBC)。
一个。对于寄生虫增殖测定,设置纯化的裂殖体至关重要,因为这可以从寄生虫中去除残留的未感染的RBC,并使我们能够检查不同宿主RBC的增殖差异(例如,比较人和猕猴RBC的侵染)。       


b。尽管应该为所有样品固定初始的寄生虫病,但获取寄生虫病的时间点0(即,用于增殖测定的平板1)仍然至关重要。这是因为起始寄生虫病的微小变化会对计算的增长率产生显着影响- 在样品之间使用不同的RBC也可以改变精确的起始寄生虫病。      


虽然此处我们使用第二个24小时时间点,但根据实验目的,可以使用其他各种时间点。24小时的时间点效果很好,因为所有寄生虫都将受到入侵并再次发展为裂殖体,使用流式细胞仪可轻松识别。不利的一面是测量了显影环和滋养体的入侵效率和生存力。较短的时间点(例如2-6小时)可用于更具体地查看入侵和早期环形成,但是这些需要非常同步和后期的裂殖体,因为在指定的时间点尚未发展为形成新环的任何裂殖体将影响数据的解释。


固定的寄生物会聚集并更快地沉淀到板的底部,否则固定和未固定的样品之间不会产生明显的影响。
测定也可以在24设置- 以1ml终体积孔板中。
如有必要,请使用移液器重新悬浮,但这会带来气泡的风险。不要涡旋)。
 


菜谱


 


固定剂:4%低聚甲醛和0.4%戊二醛
10x PBS 5毫升             


皮尔斯12.5毫升中的16%甲醛(w / v)             


ģ lutaraldehyde 125 μ 升              


H 2 O 32.5毫升             


最终体积50毫升             


LDH底物缓冲液,pH 7.5
要制备500 ml缓冲液,请混合50 ml 1 M Tris HCl(pH 8.0)和450 ml H 2 O
加入2.8克L-乳酸钠
加入1.25毫升Triton X-100
在室温下在磁力搅拌器上混合至少30分钟
制作50毫升等分试样并在-20 °C 冷冻
硝基蓝四唑(NBT)解决方案
从冰箱中取出一份50毫升的LDH缓冲液等分试样
预热至室温。
在50毫升试管中,将50毫升LDH缓冲液加入一粒NBT片剂(10 毫克)
覆盖与管铝箔,静置15分钟
不要摇动!轻轻混合
制得的溶液可以在黑暗中保持在4℃长达3周(覆盖有铝箔)
3-乙酰基吡啶腺嘌呤二核苷酸(APAD)储备溶液(10 mg / ml)
要准备10毫升的APAD储备溶液,请将100毫克的APAD溶于10毫升的蒸馏水中
存储在50分中的APAD原液μ 升在PCR管中的等分试样在-20℃下
心肌黄酶原液50单位/毫升
要制备Diaphorase储备溶液,请将1,500单位Diaphorase小瓶中的物质溶解在30 ml蒸馏水中。商店200 μ 升在-20℃下等份


完整的媒体
RPMI-1640(HEPES修改版,25 mM HEPES,不带L-谷氨酰胺,Merck),具有以下添加:


2.3 g / L碳酸氢钠(Sigma)


2 g / L葡萄糖(Sigma )


0.05克/ 升次黄嘌呤(Sigma)


5克/ 升Albumax II(Gibco)


0.3 g / L L-谷氨酰胺(10 ml 200 mM溶液,1 L培养基,默克公司)


10%(vol / vol)马血清


无菌过滤器并在4°C下储存


 


致谢


 


这项工作得到了由英国医学研究理事会和国际发展部(RWM,FM)共同资助的MRC职业发展奖(MR / M021157 / 1)的支持(Basco 等,1995; Mohring 等,2019)。 )。


 


利益争夺


 


作者宣称没有相互竞争的经济利益。


 


伦理


 


该项目,同意书和协议经LSHTM观察研究伦理委员会批准,项目编号为5520-1。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mohring, F., Rawlinson, T. A., Draper, S. J. and Moon, R. W. (2020). Multiplication and Growth Inhibition Activity Assays for the Zoonotic Malaria Parasite, Plasmodium knowlesi. Bio-protocol 10(17): e3743. DOI: 10.21769/BioProtoc.3743.
  2. Mohring, F., Hart, M. N., Rawlinson, T. A., Henrici, R., Charleston, J. A., Diez Benavente, E., Patel, A., Hall, J., Almond, N., Campino, S., Clark, T. G., Sutherland, C. J., Baker, D. A., Draper, S. J. and Moon, R. W. (2019). Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research. Elife 8: 45829.
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