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Aug 2020

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Sex-specific Separation of Plasmodium falciparum Gametocyte Populations
恶性疟原虫配子细胞群体的性别特异性分离   

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Abstract

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

Keywords: Plasmodium falciparum (恶性疟原虫), Malaria (疟疾), Gametocytes (配子体), Genetic marker (遗传标志), FACS (流式细胞荧光分选技术), Sex-specific (性别特异性), gABCG2 (gABCG2)

Background

Malaria is still one of the most important infectious diseases of humankind. Each year more than 200 million cases of malaria are reported globally, resulting in over 400,000 deaths (World Health Organization, 2019). The disease is caused by protozoan parasites of the genus Plasmodium, and P. falciparum causes the most severe disease in humans. The parasite is spread between human hosts by Anopheles mosquitoes. Male and female gametocytes (sexual blood stage parasites) are key to the transmission of malaria because only these forms can survive ingestion by the mosquito vector to complete the parasite lifecycle. Hence, understanding the biological makeup of gametocytes holds great promise to identify transmission-blocking intervention strategies.


The analysis of sexual Plasmodium stages in the laboratory poses particular challenges: only a small subset of parasites converts into sexual forms; triggers for gametocyte commitment are numerous but poorly defined; complete development of the sexual stages takes 10-12 days; and the sex ratio is female-biased (3-5:1), meaning studies on total gametocytes tend to overlook the contribution of male parasites (e.g., omics approaches or drug susceptibility studies).


Although male and female parasites can be distinguished by subtle morphological features, the enrichment of sex-specific gametocyte populations remains challenging. Previously, separate cell lines were used to obtain either male or female populations. For example, the proteome of a non-male-gametocyte-producing strain was compared to that of combined male and female gametocytes to predict the sex-specific proteome (Tao et al., 2014). Lasonder et al. (2016) later generated two independent transgenic cell lines, one with a male-specific marker (dynein heavy chain) and the other with a female-specific marker (P47), to determine the sex-specific proteome experimentally. However, when the authors introduced both tags into the same cell line, a substantial subset of the population expressed both tags, revealing that the markers were not sex-specific. Miao et al. (2017) took advantage of alpha-tubulin II (differentially expressed between sexes) to determine another sex-specific proteome. Although each of these studies contributed information on the sex-specific molecular makeup of the parasites, a more reliable and sensitive method for distinguishing male and female gametocytes could accelerate sex-specific gametocyte research.


We previously discovered a protein that is only expressed in female gametocytes (Tran et al., 2014). This molecule belongs to the ATP-binding cassette transporter family (ABCG2) and locates to a single round uncharacterized organelle in the female parasite. This protocol uses a cell line that expresses a chimeric protein consisting of gABCG2 and green fluorescent protein as a female-specific marker.


The method presented here has the advantage of sorting gametocytes from the same population, allowing for paired analysis of male and female gametocytes. The method is not only suitable for sex-specific omics approaches, but is also compatible with live-cell analysis, such as testing sex-specific drug susceptibility.


Materials and Reagents

  1. Sterile blunt end 22G needles (e.g., Livingstone, catalog number: DN22GX1.25B)

  2. 25 and 75 cm2 cell culture flasks (Corning, catalog numbers: 430168 and 430720)

  3. 175 cm2 cell culture flasks (Sarstedt, catalog number: 83.3912)

  4. Microscope slides (Hurst Scientific, catalog number: WGDFCE90)

  5. 15 and 50 ml centrifuge tubes (Corning, catalog numbers: 430791 and 430829)

  6. Disposable 500 ml bottle-top vacuum filters (Corning, catalog number: 431118) and sterile collection bottles

  7. General plastic consumables (serological pipettes, aspiration pipettes, 1.7 ml microcentrifuge tubes, 20 ml syringes, pipette tips; Sarstedt or Corning)

  8. Fresh (less than 10 days old) human red blood cells (RBCs), blood type O+ (Australian Red Cross Lifeblood)

  9. Heat-inactivated human serum pooled from at least 5 donors of the same blood type (Australian Red Cross Lifeblood)

  10. Plasmodium falciparum 3D7-gABCG2-GFP; P. falciparum 3D7 strain parasites expressing a GFP-tagged gametocyte ATP-binding cassette transporter family member 2 (gABCG2) protein (PlasmoDB accession no. PF3D7_1426500) (Tran et al., 2014)

  11. RPMI 1640 Medium, GlutaMAXTM Supplement, HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 72400120)

  12. Gentamicin 10 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15710072)

  13. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021)

  14. Hypoxanthine (Sigma-Aldrich, catalog number: H9377)

  15. Sodium hydroxide (NaOH, Sigma-Aldrich, catalog number: S5881)

  16. Milli-Q water (fresh from any available purification system)

  17. AlbuMAXTM II Lipid-Rich BSA (Thermo Fisher Scientific, GibcoTM, catalog number: 11021045)

  18. WR99210 (Jacobus Pharmaceuticals)

  19. D-Sorbitol (Sigma-Aldrich, catalog number: S6021)

  20. Methanol (for Giemsa staining) (Merck, catalog number: 1060092511)

  21. Giemsa’s solution (Merck, catalog number: 109240500)

  22. Immersion oil (Sigma-Aldrich, catalog number: 56822)

  23. N-Acetyl-D-glucosamine (GlcNAc; Carbosynth, catalog number: MA00834)

  24. Hoechst 33342 (Sigma-Aldrich, catalog number: B2261)

  25. 1× Phosphate Buffered Saline, pH 7.4 (PBS; see Recipes)

  26. 45% D-(+)-glucose (w/v) (see Recipes)

  27. 1 M NaOH (see Recipes)

  28. 200 mM hypoxanthine (see Recipes)

  29. Incomplete culture medium (ICM; see Recipes)

  30. 5% AlbuMAX II (w/v) (see Recipes)

  31. Heat-inactivated human serum (see Recipes)

  32. Complete culture medium (CCM; see Recipes)

  33. 5% D-sorbitol (w/v) (see Recipes)

  34. Complete culture medium supplemented with 50 mM GlcNAc (see Recipes)

  35. 10 mg/ml Hoechst 33342 (see Recipes)

  36. 1× PBS with 10 mM glucose (see Recipes)

  37. 10 µg/ml Hoechst 33342 in 1× PBS with 10 mM glucose (see Recipes)

Equipment

  1. Biological safety cabinet (Safemate Vision Class II, Edwards Group)

  2. Vacuum pump and liquid aspiration system (single cylinder TC501, Sparmax)

  3. 37 °C incubator (S.E.M. bench top incubator)

  4. Malaria gas mixture (1% O2, 5% CO2, 94% N2; BOC, catalog number: CCS402562G2)

  5. Water bath (WB7, Ratek)

  6. Compound microscope with 100× objective (Olympus, model: BX41)

  7. Tabletop centrifuge for 15 and 50 ml tubes (Beckman Coulter, model: Allegra X-15R)

  8. Tabletop centrifuge for microcentrifuge tubes (Thermo Scientific, model: Heraeus Pico 17)

  9. CS columns (Miltenyi Biotec, catalog number: 130-041-305) with a sterile stopcock

  10. SuperMACSTM II Separator (Miltenyi Biotec, catalog number: 130-044-104)

  11. BD FACSAriaTM Cell Sorter (BD Biosciences)

Software

  1. BD FACSDivaTM Software (BD Biosciences)

Procedure

  1. Maintenance of asexual P. falciparum

    1. Maintain asexual P. falciparum transgenic parasites (3D7-gABCG2-GFP) in complete culture medium at 4% hematocrit and less than 5% parasitemia in cell culture flasks. This transgenic strain is maintained in the presence of 2 nM WR99210. Monitor parasitemia every second day by Giemsa-stained thick smear (Maier and Rug, 2013) (Note 1). Count at least 300 cells (ideally using a blood cell counter or tally counter) to determine the culture parasitemia.

    2. Replace culture medium with complete culture medium (CCM) and split cultures to 0.2% parasitemia every second day. Flush the culture flask with malaria gas mixture of 1% O2, 5% CO2, and 94% N2 for 30 s, close the lid tightly, and incubate at 37 °C (standard culture conditions) (Maier and Rug, 2013).

    3. To generate large populations of highly purified male and female gametocytes, expand the cell culture to at least 60 ml and proceed with gametocyte production (Note 2).


  2. Production of male and female gametocytes (Figure 1)



    Figure 1. Flow-diagram outlining the timeline of the procedure


    1. Day -6. Start with 60 ml of culture at 3-5% predominantly ring parasitemia and 4% hematocrit. Perform a sorbitol synchronization as follows (Lambros and Vanderberg, 1979). Split the culture in half and transfer to two 50 ml centrifuge tubes and centrifuge at 800 × g for 7 min at room temperature (RT). Discard the supernatant and resuspend each cell pellet in 10 ml of 5% D-sorbitol. Incubate for 10 min at 37 °C. Centrifuge at 800 × g for 7 min at RT, discard the supernatant, and resuspend each cell pellet in 30 ml of CCM. Combine the suspensions from both tubes and incubate the culture under standard conditions.

    2. Day -5. Determine the parasitemia early in the day (12-18 h after synchronization) from a Giemsa-stained thick smear (Note 3). Expand the culture to 200 ml at 1% trophozoite parasitemia and 4% hematocrit in large 175 cm2 cell culture flasks. Flush the culture flask with malaria gas mixture and incubate under standard conditions.

    3. Day -4. Determine the parasitemia from a Giemsa-stained thick smear. The culture should be at 3-5% ring parasitemia. If parasitemia is lower, culture parasites for another replication cycle without allowing parasitemia to exceed 5% trophozoites. Perform sorbitol synchronization as follows (Lambros and Vanderberg, 1979). Aspirate medium carefully from the flask without disturbing the RBC layer and resuspend the cells in 30 ml of 5% D-sorbitol. Transfer the suspension into a centrifuge tube. Wash the flask with 10 ml of 5% D-sorbitol and add it to the centrifuge tube to prevent loss of cells. Incubate for 10 min at 37 °C. Centrifuge the culture at 800 × g for 7 min at RT, discard the supernatant, and resuspend the cell pellet in 30 ml of CCM. Return the suspension to the 175 cm2 cell culture flask rinsed with 30 ml of 5% D-sorbitol, top up to 200 ml with CCM, flush with malaria gas mixture, and incubate under standard conditions overnight.

    4. Day -3. Determine parasitemia from a Giemsa-stained thick smear. Expand culture to 400 ml at exactly 2% trophozoite parasitemia and 4% hematocrit in two 175 cm2 cell culture flasks (each flask containing 200 ml of culture), flush with malaria gas mixture, and incubate under standard conditions overnight.

    5. Day -2. Determine parasitemia from a Giemsa-stained thick smear. Ideally, the culture should be at 10-12% ring stage parasitemia (Note 4). Carefully remove 50 ml of medium from each cell culture flask without disturbing the cells and add 50 ml of fresh CCM (only replace 25% of the medium volume to continue to stress the culture). Flush the flasks with malaria gas mixture and incubate the cultures under standard conditions overnight.

    6. Day -1. Determine parasitemia from a Giemsa-stained thick smear. Resuspend the culture and divide equal volumes into six 175 cm2 cell culture flasks. Top up each flask to 200 ml with fresh CCM and adjust hematocrit to 4% with fresh red blood cells (this results in 1,200 ml of cell culture in 6 flasks). Flush the flasks with malaria gas mixture and incubate the cultures under standard conditions.

    7. Day 0.

      1. Perform a sorbitol synchronization (Lambros and Vanderberg, 1979) as follows. Aspirate medium carefully from each flask and resuspend the cells of each flask in 30 ml of 5% D-sorbitol. Transfer the suspension into a centrifuge tube, wash each flask with 10 ml of 5% sorbitol, and add it to the centrifuge tube to prevent loss of cells. Incubate for 10 min at 37 °C. Centrifuge the culture at 800 × g for 7 min at RT, discard the supernatant, and resuspend the cell pellet in 30 ml of CCM (resulting in a cell suspension of about 25% hematocrit).

      2. Remove asynchronous parasites at trophozoite or schizont stages and spontaneously committed gametocytes using a SuperMACS separator and CS column (Note 5).

        1. Assemble the CS column and magnetic separator according to the manufacturer’s instructions in the biological safety cabinet (Figure 2). To avoid accidental needle pricks, use a blunt end needle (22G drawing up needle, Note 6) and clip the end of the needle sheath with pliers instead of removing the sheath.



          Figure 2. Assembly of the CS column for use in the SuperMACSTM magnetic separator


        2. Sterilize the column with 25 ml of 80% ethanol (if it has previously been used) (Note 7), rinse with 25 ml of 1× PBS (pH 7.4, warmed to 37 °C), then prime with 25 ml of ICM (warmed to 37 °C) by filling the reservoir and drawing liquid through the column with the syringe. To remove air bubbles, load the syringe with ICM, reattach it to the stopcock, and flush ICM upward through the column. Draw ICM back into the syringe until the ICM level is just above the filter. Repeat flushing the column until all air bubbles are removed, then drain excess ICM through the needle until the ICM level is just above the filter. Ensure the column never runs dry from here onward (the liquid surface must always stay above the filter) as air bubbles reduce the binding surface of the column.

        3. Place column between the magnets and position a collection tube below the needle (flow resistor). Load the 25% hematocrit RBC suspension of one flask onto the column (Figure 3). Drain through the column drop-wise (adjust the flow rate to approximately 3 ml/min with the stopcock), collecting the effluent that contains uninfected red blood cells and rings committed to gametocytogenesis.



          Figure 3. CS column loaded with 25% hematocrit RBC suspension placed in the magnetic field. Stopcock is blocking downward flow through the needle. Gradually turn it clockwise to drain culture slowly.


        4. Load the column with 50 ml of CCM (warmed to 37 °C) and drain through the flow resistor into the collection tube until the effluent is no longer red.

        5. Transfer all effluent to a 175 cm2 cell culture flask and top up to 200 ml with CCM. Flush the flask with malaria gas mixture and incubate under standard conditions.

        6. Remove the column from between the magnets and rinse by loading 25 ml of ICM onto the column, drawing the liquid into the syringe, and then discarding. Repeat until the rinse is no longer brown. Fill the reservoir with 6 ml of ICM and allow it to flow through the column and needle.

        7. Repeat Steps B7b (iii-vi) until all culture flasks have been purified.

        8. Rinse the column with 25 ml of 1× PBS, 50 ml of autoclaved Milli-Q water, and 25 ml of 100% ethanol by drawing the liquid into the syringe and discarding it. Fill the reservoir with 6 ml of 100% ethanol and allow it to flow freely through the column and needle. Air-dry the column in the biological safety cabinet. Disassemble the column and stopcock and store in a sealed sterile plastic bag for reuse. Discard the used needle and syringe.

    8. Day 1. Perform a sorbitol synchronization as described on day 0 (Step B7a) but resuspend the RBC pellet of each flask (approximately 8 ml packed cell volume) in 30 ml of CCM containing 50 mM N-Acetyl-D-glucosamine (GlcNAc). Transfer the suspension to a 175 cm2 cell culture flask, top up to 200 ml with CCM containing 50 mM GlcNAc, flush the flask with malaria gas mixture, and incubate under standard conditions [Note 8; Fivelman et al. (2007)].

    9. Days 2-6. Replace the medium in the cultures daily with fresh CCM supplemented with 50 mM GlcNAc, flush the flask with malaria gas mixture, and incubate under standard conditions.

    10. Day 7. Perform a sorbitol synchronization as described on day 1 (Step B8).

    11. Day 8 (Note 9).

      1. Perform a sorbitol synchronization as described on day 0 (Step B7a), creating a cell suspension of about 25% hematocrit in ICM.

      2. Purify mature gametocytes using a SuperMACS separator and CS column. Purify and elute the gametocytes from each flask separately.

        1. Follow the same procedure as in Step B7b (i-iii). To maximize yield, reload the effluent onto the column and drain drop-wise into a waste bottle. Mature gametocytes (mostly stage IV) are retained in the column (Note 10).

        2. Load the column with 50 ml of ICM warmed to 37 °C and drain through the needle into the waste bottle until the effluent is no longer red.

        3. Remove the column from between the magnets and elute the retained gametocytes by loading the column with 25 ml of CCM and draining through the needle drop-wise into a collection tube. Transfer the effluent into a 175 cm2 cell culture flask, flush the flask with malaria gas mixture, and incubate under standard conditions.

        4. Repeat Steps B11b (i-iii) until all cultures have been purified, adding the effluent from all cultures into the same 175 cm2 cell culture flask (resulting in approximately 150 ml of gametocyte suspension).

        5. Set a small aliquot of magnet-purified culture (~1 ml) in a microcentrifuge tube and centrifuge at 2,000 × g for 1 min. Aspirate the supernatant and resuspend the pellet in 5 µl of CCM. Use this to prepare a Giemsa-stained smear to confirm the presence of gametocytes and determine their stage visually.

      3. Rinse, dry, and store the column as in Step B7b (viii).


  3. Fluorescence-activated cell sorting (FACS) of male and female gametocytes (Table 1)

    1. Prepare gametocyte samples for FACS.

      1. Resuspend magnet-enriched gametocytes. Set aside a small aliquot (~1 ml) in a microcentrifuge tube (GFP single color control). Transfer the rest of the cells to 50 ml centrifuge tubes and centrifuge at 800 × g for 10 min at RT.

      2. Discard the supernatant and resuspend all cells in 3 ml of 1× PBS with 10 mM glucose and 10 μg/ml Hoechst 33342.

      3. Incubate the sample at 37 °C for 15 min in the dark and wash twice in 1 ml of 1× PBS, with 1 min spins at 2,000 × g. After the last wash, resuspend the sample for sorting in 3 ml of 1× PBS containing 10 mM glucose.

    2. Prepare single color controls for FACS

      1. Resuspend a culture of asexual 3D7-gABCG2-GFP parasites and transfer 2× 0.5 ml of 4% hematocrit cell suspension to two microcentrifuge tubes.

      2. Set aside a small aliquot (~1 ml) of the resuspended magnet-enriched gametocyte culture.

      3. Centrifuge the above three tubes for 1 min at 2,000 × g. Discard the supernatant from the microcentrifuge tubes.

      4. To prepare the Hoechst single color control, resuspend one aliquot of asexual 3D7-gABCG2-GFP parasites in 1 ml of 1× PBS containing 10 mM glucose and 10 µg/ml Hoechst 33342. Incubate the sample at 37 °C for 15 min in the dark and wash twice in 1 ml 1× PBS with 1 min spins at 2,000 × g. After the last wash, resuspend the control in 1 ml of 1× PBS containing 10 mM glucose.

      5. Resuspend the gametocyte aliquot and the other asexual parasite aliquot in 1 ml of 1× PBS containing 10 mM glucose to prepare the GFP single color control and unstained control, respectively.


        Table 1. Single color controls and samples for FACS

        Sample    Cell culture                                                      Staining conditions
        Unstained control   Asexual 3D7-gABCG2 cell culture (~0.5 ml  aliquot)    No stain
        GFP control   3D7-gABCG2 gametocytes (~1 ml aliquot)    No stain (GFP expressed in female gametocytes)
        Hoechst 33342 control   Asexual 3D7-gABCG2 cell culture (~0.5 ml aliquot)   Stain with 10 µg/ml Hoechst 33342 in 1 ml of PBS with 10 mM glucose for 15 min in the dark at 37 °C
        Sample for sorting   3D7-gABCG2 gametocytes (~150 ml of  purified culture)   Stain with 10 µg/ml Hoechst 33342 in 3 ml of PBS with 10 mM glucose for 15 min in the dark at 37 °C


    3. Gating strategy and FACS

      1. Create a new experiment in FACS Diva. In the cytometer window, select the following channels: FSC A, H, and W; SSC A, H, and W; Pacific Blue A (or a corresponding channel to record Hoechst 33342) and Alexa Fluor 488 A (or a corresponding channel to record GFP). Create a worksheet with the dot plots (Figure 4), noting that the plots only show the events included in the parent gate.

      2. Load and run the unstained, Hoechst 33342 only, and GFP only controls. Adjust the voltages to bring the events into range (Figure 4). Adjust the single cell gates to your experimental conditions. The Hoechst only control should contain uninfected red blood cells with no staining, ring-stage infected red blood cells with low staining, and highly stained trophozoite- and schizont-infected red blood cells. Set the Hoechst intensity of the gametocyte gates to the middle population in the Hoechst single color control. The GFP single color control will contain cells with two levels of GFP intensity. Set the GFP intensity of the female gametocyte gate to match the high GFP intensity cells. The male gametocyte gate should match the GFP intensity of the unstained control.

      3. Load and run the gametocyte sample. Adjust the gating strategy if required. Prepare collection tubes filled with 2 ml of CCM at 37 °C, then start sorting cells in male and female gates into the respective collection tubes (Note 11). If multiple collection tubes are required (>10 ml), store full collection tubes at 37 °C.

      4. Check the purity of the sorted samples by analyzing a subset of each using the identical gating strategy.

      5. Since gametocytes are collected at a very low cell density, most applications of this protocol will require a final centrifugation of 1,800 × g for 10 min to pellet collected gametocytes. This protocol yields between 2 and 12 million gametocytes of each sex.



        Figure 4. Representative FACS worksheet with the gating strategy for selecting single cells. (A) and sorting gametocytes (B and C). A dot plot (B) and contour plot (C) of male (blue gate) and female (red gate) gametocytes identified by Hoechst 33342 and GFP staining.

Notes

  1. All steps should be performed using standard sterile technique with appropriate biohazard containment procedures and personal protective equipment appropriate for working with live P. falciparum parasites.

  2. Culturing can take place in dishes or flasks. As the culture volume increases, the use of flasks is advisable for ease of handling.

  3. Aim to culture at around the same time every day (within 20-24 h) to keep the availability of nutrients fairly constant.

  4. The high parasitemia induces “stress” by restriction of available nutrients and/or accumulation of metabolic waste products. This acts as a signal for gametocyte commitment.

  5. As asexual parasites and gametocytes mature in the red blood cell and digest the host hemoglobin, free heme is converted to paramagnetic hemozoin crystals. As a result, mature asexual parasites and gametocytes are retained in the column placed in a magnetic field, whereas ring-stage parasites and uninfected red blood cells pass through the magnetic column.

  6. Needle gauge size can be selected by the desired flow rate. The column manufacturer recommends 22G needle for a flow rate of 3 ml/min.

  7. Reuse column only for the same cell line to prevent any potential contamination. The column can be reused up to 5 times for large-scale cultures (1,200 ml) or until magnetic enrichment loses effectiveness.

  8. The addition of GlcNAc to the culture medium at 50 mM prevents the invasion of parasites into red blood cells and can, therefore, be used to prevent asexual parasite proliferation (Fivelman et al., 2007). This step removes all mature asexual stages still present in the culture; gametocytes will not be affected since they are not sensitive to the 5% D-sorbitol solution.

  9. Younger gametocytes at earlier developmental stages can also be harvested by reducing the days the gametocytes are in culture after commitment.

  10. Be careful to maintain the cells at 37 °C as a drop in temperature can activate mature gametocytes, resulting in gamete formation. Pre-warm all solutions to 37 °C and perform sorting at 37 °C to maintain optimal conditions. Use a slide warmer to keep flasks warm in the biosafety cabinet and a centrifuge that can be heated to 37 °C if possible.

  11. Aim for less than 20,000 events/s. If too concentrated, dilute it in PBS.

Recipes

  1. 1× PBS

    1. Dissolve 9.1 g of NaCl, 0.144 g of KH2PO4, and 0.42 g of Na2HPO4 in 800 ml of MilliQ water

    2. Adjust pH to 7.4 with HCl and top up to 1 L with water

    3. Sterilize by autoclaving

    4. Store at RT

  2. 45% D-(+)-glucose (w/v)

    1. Dissolve 45 g of D-(+)-glucose in 50 ml of warm Milli-Q water and top up to 100 ml

    2. Sterilize using a 0.22 µm filter

    3. Store 2 ml aliquots at -20 °C

  3. 1 M NaOH

    1. Dissolve 4 g of NaOH in 80 ml of Milli-Q water and top up to 100 ml

    2. Sterilize using a 0.22 µm filter

    3. Store at RT

  4. 200 mM hypoxanthine

    1. Dissolve 2.72 g of hypoxanthine in 80 ml of 1M NaOH and top up to 100 ml

    2. Sterilize using a 0.22 µm filter

    3. Store 1.2 ml aliquots at -20 °C

  5. Incomplete culture medium

    1. Add 2 ml of 45% D-(+)-glucose (w/v), 1.2 ml of 200 mM hypoxanthine, and 1 ml of 10 mg/ml gentamicin to 500 ml of RPMI 1640 Medium, GlutaMAXTM Supplement, HEPES.

    2. Store at 4 °C

  6. 5% AlbuMAX II (w/v)

    1. Dissolve 25 g of AlbuMAXTM II Lipid-Rich BSA in 500 ml of RPMI 1640 Medium, GlutaMAXTM Supplement, HEPES while shaking at 37 °C

    2. Sterilize using a 0.22 µm filter and store 37.5 ml aliquots at -20 °C

  7. Heat-inactivated human serum

    1. Pool serum from at least five donors under sterile conditions and heat them to 56 °C in a water bath while shaking occasionally to distribute heat evenly

    2. Keep the serum at 56 °C for at least 1 h

    3. Let it cool to RT. Store 12.5 ml aliquots at -20 °C

  8. Complete culture medium

    1. Add 37.5 ml of 5% Albumax II and 12.5 ml of heat-inactivated human serum to a bottle of incomplete culture medium.

    2. Store at 4 °C

  9. 5% D-sorbitol (w/v)

    1. Dissolve 50 g of D-sorbitol in 800 ml of Milli-Q water and top up to 1 L

    2. Sterilize using a 0.22 µm filter

    3. Store at 4 °C

  10. Complete culture medium supplemented with 50 mM GlcNAc

    1. Dissolve 6.1 g of GlcNAc powder in 500 ml of incomplete culture medium

    2. Sterilize using a 0.22 µm filter, then add 37.5 ml of 5% Albumax II and 12.5 ml of heat-inactivated human serum

    3. Store at 4 °C

  11. 10 mg/ml Hoechst 33342

    1. Dissolve 100 mg of Hoechst 33342 in 10 ml of Milli-Q water

    2. Sterilize using a 0.22 µm filter

    3. Store 100 µl aliquots at -20 °C

  12. 1× PBS with 10 mM glucose

    1. Add 2 ml of 45% D-(+)-glucose (w/v) to 500 ml of 1× PBS.

    2. Store at 4 °C

  13. 10 µg/ml Hoechst 33342 in 1× PBS with 10 mM glucose

    Add 5 μl of 10 mg/ml Hoechst 33342 to 4,995 µl of 1× PBS with 10 mM glucose immediately prior to use.

Acknowledgments

The authors would like to acknowledge the assistance from Dr. Harpeet Vohra and Mr. Michael Devoy with FACS and from Dr. Phuong Tran, who generated the gABCG2-GFP cell line. We are grateful to the Australian Red Cross for providing human red blood cells and serum. Funding was provided by the Australian Research Council (DP180103212). M.C.R. is supported by the Australian Government Research Training Program Scholarship and The Australian National University. The protocol is based on a method originally described in Ridgway et al. (2020).

Competing interests

The authors declare no financial or non-financial competing interests.

Ethics

The use of human red blood cells and serum was approved by the Human Ethics Committee of the Australian National University, protocol HEC#2017/351.

References

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  6. Ridgway, M.C., Shea, K.S., Cihalova, D. and Maier, A.G. (2020). Novel method for the separation of male and female gametocytes of the malaria parasite Plasmodium falciparum that enables biological and drug discovery. mSphere 5: e00671-20.
  7. Tao, D., Ubaida-Mohien, C., Mathias, D. K., King, J. G., Pastrana-Mena, R., Tripathi, A., Goldowitz, I., Graham, D. R., Moss, E., Marti, M. and Dinglasan, R. R. (2014). Sex- partitioning of the Plasmodium falciparum stage V gametocyte proteome provides insight into falciparum-specific cell biology. Mol Cell Proteomics 13: 2705-2724.
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简介

[摘要]恶性疟原虫是引起人类疟疾的单细胞真核寄生虫。食入性阶段寄生虫(称为配子体细胞)后,该寄生虫会被按蚊蚊子传播。疟疾的传播取决于寄生虫从导致疾病的无性血型转变为雌雄配子细胞。Ť他当前协议允许雌雄寄生虫的同时分离来自相同群体吨ö研究以性别-特异性方式这一关键生命周期阶段。我们已经产生了转基因恶性疟原虫 在雌性而不是雄性中表达GFP标记的寄生虫蛋白的细胞系。配子细胞的产生是应激诱导的,并且通过一系列步骤,相对于未感染的红细胞或感染了无性阶段寄生虫的红细胞,性阶段寄生虫得以富集。最后,雄性和雌性配子体由荧光分离-激活细胞分选。该方案允许从同一人群中分离出多达1200万个活体雄性和雌性寄生虫,可对其进行进一步分析。


[背景]疟疾仍然是人类最重要的传染病之一。每年全球报告超过2亿例疟疾病例,导致40万多人死亡(世界卫生组织,2019年)。本病是由属原生动物寄生虫引起的疟原虫,和恶性疟原虫引起人类最严重的疾病。这种寄生虫是由人类宿主之间传播按蚊的蚊子。男性和女性配子细胞(性血液阶段寄生虫)是疟疾传播的关键,因为只有这些形式才能通过蚊子媒介存活下来以完成寄生虫的生命周期。因此,了解配子体的生物构成了巨大希望,以确定传输-阻断干预策略。

在实验室中对性疟原虫阶段的分析提出了特殊的挑战:只有一小部分的寄生虫会转化为性形式。配子细胞定型的触发因素很多,但定义不清。性阶段的完全发展需要10到12天;性别比例为女性偏爱(3-5:1),这意味着对总配子细胞的研究往往忽略了男性寄生虫的贡献(例如,组学方法或药物敏感性研究)。

尽管男性和女性寄生虫可以通过微妙的形态特征加以区分,但是性别特异性配子细胞种群的富集仍然具有挑战性。以前,使用单独的细胞系来获得男性或女性群体。例如,将非雄性配子体产生菌株的蛋白质组与组合的雄性和雌性配子体的蛋白质组进行比较,以预测性别特异性蛋白质组(Tao等人,2014)。Lasonder等。(2016)后产生两个独立的转基因细胞系,一个与特定的雄性标记(动力蛋白重链)和另一个具有母-特异性标记物(P47) ,通过实验来确定的性别特异性蛋白质组。然而,当笔者介绍了这两个标签到相同的细胞系,大幅的的子群中表达这两个标签,灵兽荷兰国际集团的标志不是性别特异性。苗等。(2017年)趁着阿尔法-微管蛋白II (两性之间的差异表达),确定另一性别特异性蛋白质组。尽管这些研究中的每一项都提供了有关寄生虫性别特异性分子组成的信息,但是一种更可靠,更灵敏的区分雄性和雌性配子细胞的方法可以加快性别特异性配子细胞的研究。

我们之前发现了一种仅在雌性配子体细胞中表达的蛋白质(Tran等,2014)。此分子属于ATP结合盒转运蛋白家族(ABCG2),位于雌性寄生虫中的单个圆形未表征的细胞器中。该方案使用一种细胞系,该细胞系表达由gABCG2和绿色荧光蛋白组成的嵌合蛋白作为雌性特异性标记。

此处介绍的方法的优点是可以对来自同一种群的配子体细胞进行分类,从而可以对雄性和雌性配子体进行配对分析。该方法不仅适用于性别特定的组学方法,而且与活细胞分析兼容,例如测试性别特定的药物敏感性。

关键字:恶性疟原虫, 疟疾, 配子体, 遗传标志, 流式细胞荧光分选技术, 性别特异性, gABCG2



材料和试剂


无菌平端22G针头(例如,利文斯通,目录号:DN22GX1.25B)
25和75 cm 2细胞培养瓶(Corning,目录号s :430168和430720)
175 cm 2细胞培养瓶(Sarstedt ,目录号:83.3912)
显微镜载玻片(Hurst Scientific,目录号:WGDFCE90)
15和50 ml离心管(Corning,目录号s :430791和430829)
一次性500毫升瓶顶真空过滤器(Corning,目录号:431118)和无菌收集瓶
一般塑料消耗品(血清移液管,吸液移液管,1.7 m l微量离心管,20 m l注射器,移液管头;Sarstedt或Corning)
新鲜的(不到10天)人血红细胞(RBC),血型为O + (澳大利亚红十字会的生命线)
从至少5种相同血型的供体中收集热灭活的人血清(澳大利亚红十字会的生命线)
恶性疟原虫3D7-gABCG2-GFP ; 表达GFP标签的配子配体ATP结合盒转运蛋白家族成员2(gABCG2)蛋白的恶性疟原虫3D7菌株寄生虫(PlasmoDB登录号PF3D7_1426500)(Tran等,2014)
RPMI 1640 Medium,GlutaMAX TM Supplement,HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:72400120)
庆大霉素10 mg / ml(Thermo Fisher Scientific,Gibco TM ,目录号:15710072)
D-(+)-葡萄糖(Sigma - Aldrich,目录号:G7021)
次黄嘌呤(Sigma - Aldrich,目录号:H9377)
氢氧化钠(NaOH,Sigma - Aldrich,目录号:S5881)
Milli - Q水(来自任何可用的净化系统的新鲜水)
AlbuMAX TM II富脂BSA(Thermo Fisher Scientific,Gibco TM ,目录号:11021045)
WR99210(Jacobus制药)
D-山梨糖醇(Sigma - Aldrich,目录号:S6021)
甲醇(用于吉姆萨染色)(默克,目录号:1060092511)
Giemsa的解决方案(Merck,目录号:109240500)
浸油(Sigma - Aldrich,目录号:56822)
N-乙酰基-D-葡萄糖胺(GlcNAc ; Carbosynth ,目录号:MA00834)
Hoechst 33342(Sigma - Aldrich,目录号:B2261)
1×磷酸盐缓冲盐水,pH 7.4(PBS ;参见配方)
45%D-(+)-葡萄糖(w / v)(参见食谱)
1 M NaOH (请参阅食谱)
200 mM次黄嘌呤(参见食谱)
不完整的培养基(ICM ;请参阅食谱)
5%AlbuMAX II(w / v)(参见食谱)
热灭活的人血清(请参见食谱)
完全培养基(CCM ;请参阅食谱)
5%D-山梨糖醇(w / v)(参见食谱)
完整培养基,添加50 mM GlcNAc (请参阅食谱)
10 mg / ml Hoechst 33342 (请参阅食谱)
1 × PBS,含10 mM葡萄糖(请参阅食谱)
含10 mM葡萄糖的1×PBS中的10 µg / ml Hoechst 33342 (请参阅食谱)


设备


生物安全柜(爱德华兹集团的Safemate Vision Class II)
真空泵和液体抽吸系统(单缸TC501,Sparmax )
37°C恒温箱(SEM台式恒温箱)
疟疾气体混合物(1%氧气2 ,5%CO 2 ,94%N 2 ; BOC,目录号:CCS402562G2)
水浴(WB7,Ratek )
用100×物镜化合物显微镜(Olympus,米Odel等:BX41)
台式离心机15个50毫升管(Beckman Coulter公司,米Odel等:Allegra的X-15R)
台式离心机,用于微量离心管(Thermo Scientific,型号:Heraeus Pico 17)
带有无菌旋塞阀的CS色谱柱(Miltenyi Biotec ,目录号:130-041-305)
SuperMACS TM II分离器(Miltenyi Biotec ,目录号:130-044-104)
BD FACSAria TM细胞分选仪(BD Biosciences)


软件


BD FACSDiva TM软件(BD Biosciences)


程序


无性恶性疟原虫的维持
1.在完全培养基中,在细胞培养瓶中以4%的血细胞比容和少于5%的寄生虫血症维持无性恶性疟原虫转基因寄生虫(3D7-gABCG2-GFP)。该转基因菌株在2nM WR99210的存在下得以维持。监视器原虫每隔一天用姬姆萨染色涂片厚(迈尔和地毯,2013年)(注1) 。计数至少300个细胞(最好使用血细胞计数器或计数计数器)以确定培养物中的寄生虫血症。     

2.用完全培养基(CCM)代替培养基,并每隔2天将培养物分裂为0.2%的寄生虫血症。齐平的疟疾气体混合物中的培养瓶1%氧气2 ,5%CO 2 ,和94%N 2 30秒,关闭盖紧紧,孵育在37℃下(标准培养条件)(迈尔和地毯,2013 )。     

3.为了产生大量高度纯化的雄性和雌性配子细胞,将细胞培养物扩展到至少60 ml,然后继续配子细胞的生产(注2)。     



雄配子和雌配子的产生(图1)


图1.流程图概述了过程的时间表


第六天 从60 ml培养液开始,培养液的3-5%主要是环寄生性贫血和4%的血细胞比容。按如下步骤进行山梨醇同步化处理(Lambros和Vanderberg ,1979)。将培养物分成两半,转移到两个50 ml离心管中,在室温(RT)下以800 × g离心7分钟。丢弃上清液,并将每个细胞沉淀重悬于10 ml的5%D-山梨糖醇中。在37°C下孵育10分钟。在室温下以800 × g离心7分钟,弃去上清液,然后将每个细胞沉淀重悬于30 ml CCM中。合并两个试管的悬浮液,并在标准条件下孵育培养物。
第-5天。从吉姆萨(Giemsa)染色的浓稠涂片(注3)的清晨(同步后12-18 h )确定寄生虫血症。在1个175 cm 2大细胞培养瓶中,在1%滋养体寄生虫和4%血细胞比容下将培养液扩展至200 ml 。用疟疾气体混合物冲洗培养瓶,并在标准条件下孵育。
第四天 从吉姆萨染色浓密的涂片确定寄生虫病。培养应在3-5%的环寄生虫病中进行。如果寄生虫血症较低,则培养寄生虫进行另一个复制周期,而不允许寄生虫毒素超过5%的滋养体。如下进行山梨醇同步化(Lambros和Vanderberg ,1979)。小心吸出培养基从烧瓶中,而不会干扰RBC层和重悬细胞于30ml 5%d的-山梨糖醇。将悬浮液转移到离心管中。用10毫升5%的d洗烧瓶-山梨糖醇,并将其加入到离心管中,以防止细胞的损失。在37°C下孵育10分钟。在室温下将培养物以800 × g离心7分钟,弃去上清液,然后将细胞沉淀重悬于30 ml CCM中。将悬浮液放回175 cm 2细胞培养瓶中,用30 ml 5%D-山梨醇冲洗,用CCM加至200 ml,用疟疾气体混合物冲洗,并在标准条件下孵育过夜。
第-3天。从吉姆萨染色浓密的涂片中确定寄生虫病。在两个175 cm 2细胞培养瓶(每个瓶中包含200 ml培养物)中,在2%滋养体寄生虫和4%血细胞比容下将培养物扩展至400 ml ,用疟疾混合气体冲洗,并在标准条件下孵育过夜。
第-2天。从吉姆萨染色浓密的涂片中确定寄生虫病。理想情况下,培养应处于10-12%的环形阶段寄生虫血症(注4)。小心地取出50毫升的从每个细胞培养瓶培养基而不扰乱细胞和加入50mL的新鲜CCM(只更换介质体积的25%继续强调培养物)。用疟疾气体混合物冲洗烧瓶,并在标准条件下孵育培养物过夜。
第-1天。从吉姆萨染色浓密的涂片中确定寄生虫病。重悬培养并将等体积分成六个175 cm 2细胞培养瓶。用新鲜的CCM加满每个烧瓶至200 ml,并用新鲜的红细胞将血细胞比容调节至4%(这将在6个烧瓶中产生1200 ml的细胞培养物)。用疟疾气体混合物冲洗烧瓶,并在标准条件下孵育培养物。
第0天。
如下进行山梨醇同步化(Lambros和Vanderberg ,1979)。从每个烧瓶中小心吸出培养基,并将每个烧瓶中的细胞重悬于30 ml的5%D-山梨糖醇中。将悬浮液转移到离心管中,用10 ml的5%山梨糖醇洗涤每个烧瓶,并将其添加到离心管中以防止细胞丢失。在37°C下孵育10分钟。在室温下将培养物以800 ×g离心7分钟,弃去上清液,然后将细胞沉淀重悬于30 ml CCM中(产生约25%血细胞比容的细胞悬液)。
使用SuperMACS分离器和CS色谱柱除去滋养体或裂殖体阶段的异步寄生虫和自发定殖的配子细胞(注5)。
组装的根据制造商的在生物安全柜的指令(图2)CS柱和磁分离器。为避免意外刺针,请使用钝的末端针头(22G拔起针头,注意6),并用钳子夹住针头护套的末端,而不要取下护套。




图2.用于SuperMACS TM磁选机的CS色谱柱的组装


用25ml的80%乙醇(如果消毒柱它先前已被使用)(注7),冲洗,用25ml的1×PBS的(pH值7.4 ,加热到37 ℃下),然后素用25ml ICM的(通过将储液器注满并用注射器将液体从色谱柱中吸出,将温度升至37°C )。要除去气泡,请在注射器上装入ICM,将其重新安装到旋塞阀上,然后向上冲洗ICM穿过色谱柱。绘制ICM回注射器中,直到该ICM水平正好处于过滤器上方。重复冲洗该柱,直到所有气泡被除去,然后通过针排出多余的ICM直到所述ICM水平仅仅是过滤器上方。由于气泡会减少色谱柱的结合表面,因此请确保色谱柱从此处开始永远不会干dry(液体表面必须始终保持在过滤器上方)。
将柱子放在磁铁之间,并在针(流动电阻器)下方放置一个收集管。将一个烧瓶的25%血细胞比容RBC悬浮液加载到色谱柱上(图3 )。逐滴通过色谱柱排干(用旋塞阀将流速调节至约3 ml / min),收集流出液,该流出液包含未感染的红血球和参与配子形成的环。


图3.在磁场中装有25%的红细胞比容RBC悬浮液的CS色谱柱。旋塞阀阻止向下流过针头。逐渐将其顺时针旋转以缓慢排出培养液。


将50 ml CCM (加热到37°C )装满色谱柱,并通过流动电阻器将其排放到收集管中,直到流出物不再是红色为止。
将所有流出物转移到175 cm 2的细胞培养瓶中,并用CCM加满200 ml。用疟疾气体混合物冲洗烧瓶,并在标准条件下孵育。
从磁体之间移出色谱柱,并通过将25 ml ICM加载到色谱柱上,将液体吸入注射器中,然后丢弃进行冲洗。重复直到冲洗液不再呈褐色。用6 ml ICM填充储槽,使其流过色谱柱和针头。
重复小号TEPS B7 B(三-六),直到所有的培养瓶已被纯化。
冲洗用25ml的1×PBS,将50ml的柱的高压灭菌的中号ILLI - Q水,通过将液体到注射器和废弃和25ml 100%乙醇它。用6 ml 100%乙醇填充储液槽,使其自由流过色谱柱和针头。风干生物安全柜中的色谱柱。拆卸色谱柱和旋塞阀,然后将其存储在密封的无菌塑料袋中以备重复使用。丢弃用过的针头和注射器。
每日1作为第0天(描述执行山梨醇同步小号TEP乙7一),但重悬每个烧瓶中的RBC沉淀在30ml含50 CCM(约8毫升积细胞体积)毫N-乙酰基d葡糖胺(GlcNAc )。将悬浮液转移至175 cm 2的细胞培养瓶中,用含50 mM GlcNAc的CCM加满200 ml,用疟疾气体混合物冲洗培养瓶,并在标准条件下孵育[注8; Fivelman等。(2007)] 。
天小号2 - 6.更换该培养物中的日常用补充有50新鲜CCM介质毫的GlcNAc ,冲洗以疟疾气体混合物中的烧瓶中,在标准条件下孵育。
如在第1天(天描述7.执行山梨醇同步小号TEP乙8)。
第8天(注9)。
如在第0天(描述执行山梨醇同步小号TEP乙7a)中,创建约25%的细胞悬浮液的血细胞比容在ICM。
使用SuperMACS分离器和CS柱纯化成熟的配子细胞。分别纯化和洗脱每个烧瓶中的配子细胞。
按照相同的程序,在小号TEP乙7B(我- ⅲ)。为了最大程度地提高产量,将废水重新装到色谱柱上,然后逐滴排放到废液瓶中。成熟的配子细胞(大部分为IV期)保留在色谱柱中(注10)。
在柱子上加入50 ml加热至37°C的ICM,并通过针头排入废液瓶,直到流出液不再呈红色。
从磁体之间移走色谱柱,并通过向色谱柱中加载25 ml CCM并通过针头滴入收集管的方式将其洗脱,从而洗脱保留的配子细胞。将废水转移到175 cm 2的细胞培养瓶中,用疟疾气体混合物冲洗该瓶,并在标准条件下孵育。
重复小号TEPS B11B(我- III ),直到所有的培养物已被纯化,加入从所有培养物的流出物进入相同175厘米2细胞培养瓶(得到约imately 150毫升配子体悬浮液)。
在微量离心管中放置一小部分经过磁铁纯化的培养物(约1 ml),并以2,000 × g离心1分钟。吸出上清,重悬在5颗粒μ升CCM的。使用此准备吉姆萨-染色涂片确认配子体的存在,并确定他们的舞台视觉。
漂洗,干燥,和列存储为小号TEP乙7B(VIII)。


男女配子细胞的荧光激活细胞分选(FACS)(表1)
      准备配子体样品小号的FACS。
重悬富含磁铁的配子细胞。在微量离心管(GFP单色对照)中放置一小份(〜1 ml )。将其余的细胞转移到50 ml离心管中,在RT下以800 ×g离心10分钟。
丢弃上清液并将所有细胞重悬于3 ml的1×PBS中,其中含10 mM葡萄糖和10μg / ml Hoechst 33342。
在黑暗中于37°C孵育样品15分钟,然后在1 ml的1×PBS中洗涤两次,并在2,000× g的条件下旋转1分钟。最后一次洗涤后,将样品重悬以在3 ml的含有10 mM葡萄糖的1x PBS中分选。
      准备用于FACS的单色控件
                                          重悬无性3D7-gABCG2-GFP寄生虫的培养物,并将2×0.5 ml的4%血细胞比容细胞悬液转移到两个微量离心管中。
保留一小部分(〜1 ml)重悬浮的富含磁铁的配子细胞培养物。
将上述三个试管以2,000 ×g离心1分钟。弃去微量离心管的上清液。
要制备Hoechst单色对照,将等分的无性3D7-gABCG2-GFP寄生虫重悬于1 ml的含有10 mM葡萄糖和10 µg / ml Hoechst 33342的1x PBS中。在37°C下将样品孵育15分钟。深色,在2,000 × g中以1分钟旋转1分钟的1×PBS中洗涤两次。最后一次洗涤后,将对照重悬于1 ml的含有10 mM葡萄糖的1x PBS中。
重悬配子体等分试样并在1ml的1×PBS的含有10mM葡萄糖制备GFP单其它无性寄生虫等分颜色控制和未染色的对照,分别。


表1.用于FACS的单色控件和样本


      门控策略和FACS
在FACS Diva中创建一个新实验。在流式细胞仪窗口中,选择以下途径:FSC A,H ,和W; SSC A,H ,和W; 太平洋蓝A(或用于记录Hoechst 33342的相应通道)和Alexa Fluor 488 A(或用于记录GFP的相应通道)。创建一个带有点图的工作表(图4 ),注意这些图仅显示父门中包含的事件。
加载并运行未染色的(仅Hoechst 33342 )和仅GFP的对照s 。调整电压以使事件进入范围(图4 )。根据您的实验条件调整单细胞门。赫司特仅控制应该含有未感染的红细胞没有染色,环级感染红血细胞具有低染色,和高度染色trophozoite-和裂殖体感染的红细胞。在Hoechst单色控件中,将配子细胞门的Hoechst强度设置为中等种群。GFP单色对照将包含具有两个GFP强度水平的细胞。设置雌性配子体细胞门的GFP强度,以匹配高GFP强度的细胞。雄性配子体细胞门应与未染色对照的GFP强度匹配。
加载并运行配子细胞样品。如果需要,调整选通策略。准备在37 °C下装有2 ml CCM的收集管,然后开始将雄性和雌性浇口中的细胞分选到各个收集管中(注11)。如果需要多个收集管(> 10 ml),请将完整的收集管存放在37°C下。
检查的纯度的,通过使用相同的门控策略分析每个子集排序的样本。
由于配子细胞是在非常低的细胞密度下收集的,因此该方案的大多数应用都需要将最终离心力为1,800 × g,持续10分钟,以沉淀收集的配子细胞。该方案可产生每种性别2至1200万个配子细胞。




图4. ř具有代表性FACS工作表用于选择单个细胞的门控策略。(A)和分类配子体(B和C)。通过Hoechst 33342和GFP染色鉴定的雄性(蓝门)和雌性(红门)配子细胞的点图(B)和轮廓图(C)。                                                     



笔记


所有步骤均应使用标准的无菌技术,适当的生物危害遏制程序以及适用于恶性疟原虫活体寄生虫的个人防护设备执行。
培养可以在盘子或烧瓶中进行。随着培养物体积的增加,建议使用培养瓶以便于处理。
力争每天大约在同一时间(20-24小时内)进行培养,以保持营养素的可用性相当稳定。
高寄生物血症通过限制可用营养素和/或代谢废物的积累而引起“压力”。这充当配子体定型的信号。
作为无性寄生虫和配子体在红血细胞成熟和消化主机血红蛋白,自由下摆ë转化为顺磁性疟原虫色素晶体。结果,成熟的无性寄生虫和配子细胞保留在放置在磁场中的柱子中,而环状阶段的寄生虫和未感染的红血球则通过该柱子。
针规的大小可以通过所需的流速来选择。色谱柱制造商建议使用22G针,流速为3 ml / min。
仅可将色谱柱重复用于同一细胞系,以防止任何潜在的污染。该色谱柱最多可重复使用5次用于大规模培养(1,200 ml ),或者直到磁富集失去效力为止。
加入的GlcNAc到的以50mM防止寄生虫侵入红细胞和罐培养基,因此,可用于防止无性寄生虫增殖(Fivelman等人,2007)。这个步骤去除了文化中仍然存在的所有成熟的无性恋阶段。配子体细胞不会受到影响,因为它们对5%D-山梨糖醇溶液不敏感。
还可以通过减少承诺后配子细胞在培养中的天数来收获较早发育阶段的较年轻配子细胞。
注意将细胞保持在37°C,因为温度下降会激活成熟的配子细胞,导致配子形成。将所有溶液预热至37°C,并在37°C下进行分选以保持最佳条件。使用载玻片加热装置将烧瓶放在生物安全柜中,并在可能的情况下将其加热到37 °C的离心机。
瞄准少于20,000个事件/秒。如果浓度太高,则用PBS稀释。


菜谱


1×PBS
溶解9.1克的氯化钠,0.144克的KH 2 PO 4 ,和0.42克的的Na 2 HPO 4在800毫升的MilliQ水
用HCl调节pH值至7.4,并用水补足至1 L                           
高压灭菌
存放在RT
45%D-(+)-葡萄糖(w / v)
将45 g D-(+)-葡萄糖溶解在50 ml温暖的Milli - Q水中,加满100 ml
使用0.22 µm过滤器灭菌
在-20 °C下储存2 ml等分试样
1 M氢氧化钠
将4 g NaOH溶解在80 ml Milli - Q水中,加满100 ml
使用0.22 µm过滤器灭菌
存放在RT
200 mM次黄嘌呤
将2.72克次黄嘌呤溶于80毫升1M氢氧化钠中,加至100毫升
使用0.22 µm过滤器灭菌
在-20°C下储存1.2 ml等分试样
不完整的培养基
甲DD2毫升45%d的- (+) -葡萄糖(W / V),1.2毫升的200mM次黄嘌呤,和1ml 10mg / ml的庆大霉素吨ö500毫升RPMI 1640培养基中,GlutaMAXTM补编,HEPES。
储存在4°C
5%的AlbuMAX II(w / v)
将25 g AlbuMAXTM II高脂BSA溶于500 ml RPMI 1640培养基,GlutaMAX TM Supplement,HEPES中,同时在37°C摇动
使用0.22 µm过滤器灭菌,并在-20°C下储存37.5 ml等分试样
热灭活的人血清
在无菌条件下收集至少五名供体的血清,并在水浴中将其加热至56°C,同时不时摇动以均匀分布热量
保持血清在56°C至少1小时
让其冷却至RT 。在-20°C下储存12.5 ml等分试样
完整的培养基
甲DD37.5毫升5%Albumax II和12.5 ml热的-灭活人血清吨○瓶不完全培养基。
储存在4°C
5%D-山梨糖醇(w / v)
将50克D-山梨糖醇溶解在800毫升Milli - Q水中,加满1升水
使用0.22 µm过滤器灭菌
储存在4°C
完整培养基,添加50 mM GlcNAc
将6.1 g GlcNAc粉末溶解在500 ml不完全培养基中
消毒使用0.22微米的过滤器,再加入37.5毫升5%Albumax II和12.5毫升热-灭活的人血清
储存在4°C
10毫克/毫升赫斯特33342
将100毫克Hoechst 33342溶于10毫升Milli - Q水中
使用0.22 µm过滤器灭菌
在-20°C下储存100 µl等分试样
1 ×含10 mM葡萄糖的PBS
甲DD2毫升45%d的- (+) -葡萄糖(W / V)吨Ö500毫升的1×PBS 。
储存在4°C
含10 mM葡萄糖的1×PBS中的10 µg / ml Hoechst 33342
加入5微升的10毫克/毫升的Hoechst 33342至4995微升1×PBS与10mM葡萄糖在使用前立即。


致谢


作者要感谢来自博士的协助Harpeet沃赫拉和迈克尔先生Devoy用流式细胞仪及从PHUONG陈医生,谁产生的gABCG2-GFP的细胞系。我们感谢澳大利亚红十字会提供人类红细胞和血清。资金由澳大利亚研究理事会(DP180103212)提供。MCR得到了澳大利亚政府研究培训计划奖学金和澳大利亚国立大学的支持。该协议基于Ridgway等人最初描述的方法。(2020年)。


利益争夺


作者声明没有任何金融或非金融竞争利益。


伦理


人类红细胞和血清的使用已得到澳大利亚国立大学人类伦理委员会的批准,规程为HEC#2017/351。


参考


1. Fivelman ,Q 。大号。,麦克罗伯特,L 。,夏普,S 。,泰勒,C 。Ĵ 。,Saeed ,M 。,斯威尔士,C 。一。,萨瑟兰,C 。Ĵ 。和贝克,D 。答(2007)。改进的同步生产恶性疟原虫配体细胞。Mol Biochem Parasitol 154:11 9 -1 2 3。     

2. Lambros,C.和Vanderberg,J . P. (1979 )。培养中恶性疟原虫红细胞阶段的同步。Ĵ寄生虫学65 :418 - 4 20。     

3. Lasonder ,E 。,Rijpma ,S 。[R 。,van Schaijk ,B 。Ç 。大号。,Hoeijmakers ,W 。一。中号。,Kensche ,P 。[R 。,Gresnigt ,M 。小号。,Italiaander ,A 。,沃斯,M 。w ^ 。,Woestenenk ,R 。,Bousema ,T 。,梅尔,G 。[R 。,Khan ,S . 中号。,Janse ,C . Ĵ 。,Bártfai ,R 。和Sauerwein ,R 。W.(2016年)。恶性疟原虫配子细胞的转录组学和蛋白质组学综合分析:对性别特定过程和翻译抑制的分子了解。核酸研究44:6087 - 6101。     

4. Maier ,A 。摹。和Rug ,M。(2013)。体外培养恶性疟原虫红细胞阶段。方法分子生物学923:3 - 15 。     

5.苗,J 。陈,ž 。王,ž 。,Shrestha ,S . 李,X 。李,[R 。和Cui ,L.(2017年)。从成熟的男性和女性配子细胞的蛋白质组揭示了人类疟原虫的性别特异性生物学。分子细胞蛋白质组学16:537 - 551。     

6.李奇微,M. C.,乳木果,KS,Cihalova ,D.和麦尔,AG(2020)。分离疟原虫恶性疟原虫的男性和女性配子细胞的新方法,可以实现生物学和药物发现。mSphere 5:e00671-20。     

7.陶,D 。,Ubaida-Mohien ,C 。,马蒂亚斯(Mathias),D 。ķ 。,金,J 。G. ,Pastrana-Mena ,R 。,Tripathi ,A 。,哥德威兹,我。,格雷厄姆,D 。[R 。,莫斯,E 。,Marti ,M 。和Dinglasan ,R 。R.(2014年)。恶性疟原虫V期配子细胞蛋白质组的性别划分提供了对恶性疟原特异性细胞生物学的见识。分子细胞蛋白质组学13:2705 - 2724。     

8. Tran,P. N.,Brown,S. H.,Mitchell,T. W.,Matuschewski,K.,McMillan,P. J.,Kirk,K.,Dixon,M.W .和Maier,A 。G.(2014)。雌性配子体特异性ABC转运蛋白在疟原虫的脂质代谢中发挥作用。Nat Commun 5 (4773):1-13。     

9.世界卫生组织(2019年)。《 2019年世界疟疾报告》。      
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引用:Ridgway, M. C., Cihalova, D. and Maier, A. G. (2021). Sex-specific Separation of Plasmodium falciparum Gametocyte Populations. Bio-protocol 11(11): e4045. DOI: 10.21769/BioProtoc.4045.
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