Materials and Reagents

1. Rosette-forming iRBCs
   1. Culture of rosette-forming parasites at mature stages (trophozoite to early schizont stages, 5-10% parasitaemia, 5% haematocrit)
      
   2. Parasite clonal lines 3D7/PF13, Palo Alto 89F5 VarO, IT4/R29 or patient isolates
      
      
2. Culture medium reagents
   1. Malaria non-immune human AB⁺ serum (pool of 3-5 different donors)
      
   2. RPMI 1640 medium with L-glutamine and 25 mM HEPES (500 ml) (Life Technologies, Gibco^®, catalog number: 52400 )
      
   3. 10 mM Hypoxanthine solution (100x) (C.C.Pro, catalog number: Z-41-M )
      
   4. Gentamicin solution (50 mg/ml) (Sigma-Aldrich, catalog number: G1397 )
      
      
3. Other materials
   1. Non pyrogenic sterile polystyrene, Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap (75 cm², Corning Incorporated, catalog number: 430720 ; 25 cm²,  Corning Incorporated, catalog number: 430168 , for 20 and 5 ml culture medium, respectively)
      
   2. Ice cold Ficoll (Lymphoprep^TM, density 1.077 g/ml) (Abcys, catalog number: 1114545 )
      
   3. Dextran sulfate sodium salt from Leuconostoc spp, MW > 500,000 (Sigma-Aldrich, catalog number: D6001 )
      
   4. Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3393 )
      
   5. Fucoidan from Fucus vesiculosus (Sigma-Aldrich, catalog number: F5631 )
      
   6. Hoechst 33342, 10 mg/ml solution in water (Life Technologies, Molecular Probes^®, catalog number: H-3570 )
      
   7. Sterile distilled water RNase DNase free (Life Technologies, Gibco^®, catalog number: 10977-035 )
      
   8. Mouse, rat or rabbit immune and pre-immune polyclonal sera
      
   9. Monoclonal antibodies (mAbs) reacting with the surface of rosette-forming iRBCs
      
   10. Serum or plasma samples from individuals living in a malaria endemic region
   11. Serum or plasma samples from malaria non-immune individuals
       Note: All plasma samples must be collected using an anticoagulant other than heparin (heparin disrupts rosettes of multiple strains at the concentration used for anticoagulant activity. Convenient anticoagulants are EDTA [ethylene diamine tetraacetic acid] or sodium citrate). Store at -20 °C or -80 °C until use.
       Note: Abs are home made or collected from patients.
       

Equipment

1. 5 ml polystyrene round-bottom sterile tube (BD Biosciences, Falcon^®, catalog number: 352058 )
   
2. Sterile laboratory vacuum filter 0.22 µm (Stericup^® Filter Units Millipore, catalog number:  051446 )
   
3. Sterile polypropylene conical centrifuge tubes (15 ml, BD Biosciences, Falcon^®, catalog number: 352097 ;  50 ml, BD Biosciences, Falcon^®, catalog number: 352098 )
   
4. Sterile disposable serological 1 ml aspiration pipette (Dominique Dutscher, catalog number:  999079 )
   
5. Professional gloves (e.g. Kimtech Sterling Nitrile gloves, Kimberley-Clark Professional, catalog number: 99211 , or Satin Plus gloves Kimberley-Clark Professional, catalog number: SP2330E or 2220E )
   
6. 1.4 ml matrix round bottom non sterile tubes (Thermo Fisher Scientific, catalog number:  10630784 )
   
7. Microscope glass slides (Thermo Fisher Scientific, catalog number: 10090431 ) and cover slips 22 x 22 mm (Thermo Fisher Scientific, catalog number: 11728691 )
   
8. Centrifuge with a swing bucket rotor (Thermo Fisher Scientific Heraeus Multifuge 3SR+ centrifuge )
   
9. Centrifuge Eppendorf 5702 with an A-4-38 swing bucket rotor (Thermo Fisher Scientific, catalog number: 05-400-318)
   
10. Vacuum pump (ILMVAC Biovac, model: 104 )
    
11. Fluorescence microscope with UV-light and 40x or 100x magnification (Leica fluorescence microscope DM5000B, HP Plan ocular 10 x 22 507897, HCX Plan 100x Oil, HCXPL 40x PH2)
    
12. Incubator at 37 °C in continuous gazing: 5% O₂, 5% CO₂ and 90% N₂ (Thermo Fisher Scientific Binder incubator, model: CB210 )
    
13. Laminar flow class II, type A2 biological safety cabinet (e.g. Thermo Fisher Scientific HeraSafe, catalog number: 13-998-002 )
    

Procedure