Plasmodium falciparum Rosette Formation Assay

Materials and Reagents

1. Human red blood cells and parasitized red blood cells
   1. Fresh human red blood cells (RBCs) collected on anticoagulant, preferably citrate-phosphate-dextrose (CPD). Avoid use of heparin, as most rosettes are heparin-sensitive. If fresh RBCs (collected during the previous 24 h) are not available, use a sample stored for less than 2 weeks at 4 °C.
      
   2. P. falciparum parasitized red blood cells collected by veinipuncture from patients in the presence of CPD anticoagulant
      
   3. Laboratory lines of P. falciparum parasites: 3D7 clone obtained from MR4 or rosetting clones such as Palo Alto 89F5 VarO, IT4/R29, 3D7/PF13 (Vigan-Womas et al., 2011) or IT4/FCR3S1.2
      
      
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. Hypoxanthine solution 100x (10 mM) (C.C.Pro, catalog number: Z-41-M )
   4. Gentamicin solution (50 mg/ml) (Sigma-Aldrich, catalog number: G1397 )
      
   5. Human AB⁺ serum (final concentration 10%)
   6. Complete culture medium (see Recipes)
      
      
3. Other materials
   1. Non pyrogenic sterile polystyrene, Rectangular Canted Neck Cell Culture Flask with Plug Seal Cap (Corning Incorporated 75 cm², catalog number: 430720 , 25 cm², catalog number: 430168 , for 20 and 5 ml of culture medium, respectively)
      
   2. 5 ml polystyrene round-bottom sterile tube (BD Biosciences, Falcon^®, catalog number: 352058 )
      
   3. Sterile laboratory vacuum filter 0.22 μm (Stericup^® Filter Units Millipore, catalog number: 051446 )
      
   4. Sterile polypropylene conical centrifuge tubes (BD Biosciences, Falcon^® 15 ml) (BD Biosciences,  catalog number: 352097 ), (Falcon 50 ml) (BD Biosciences, catalog number: 352098 )
      
   5. Sterile disposable serological 1 ml aspiration pipette (Dominique Dutscher, catalog number: 999079 )
      
   6. 1.4 ml matrix round bottom non sterile tubes (Thermo Fisher Scientific, catalog number: 10630784 )
      
   7. 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 )
      
   8. Microscope glass slides (Thermo Fisher Scientific, catalog number: 10090431 ) and cover slips 22 x 22 mm (Thermo Fisher Scientific, catalog number: 11728691 )
      
   9. Ice cold Ficoll (Lymphoprep^_TM, density 1.077 g/ml) (Abcys, catalog number: 1114545 )
      
   10. Dextran sulfate sodium salt from Leuconostoc spp, MW > 500,000 (Sigma-Aldrich, catalog number: D6001 )
       
   11. Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H3393 )
       
   12. Hoechst 33342, 10 mg/ml solution in water (Molecular Probes, catalog number: H-3570 )
       
   13. PKH67 green fluorescent cell linker (Sigma-Aldrich, catalog number: MINI-67 )
       
   14. PKH26 red fluorescent cell linker (Sigma-Aldrich, catalog number: MINI-26 )
       
   15. Foetal bovine serum (Sigma-Aldrich, catalog number: F2442 )
       
   16. Sterile distilled water RNase DNase free (Life Technologies, Gibco^®, catalog number: 10977-035 )
       
   17. Giemsa staining reagent: Giemsa R solution (RAL Diagnostics, catalog number: 320310-0500 )
       
   18. Giemsa buffer tablet pH 7.2 (Merck KGaA, catalog number: 1.09468.0100 )
       
   19. Methanol GPR Rectapur BDH Proloabo (catalog number: 20846.292 )
       
   20. Sterile distilled water
   21. Stock solutions of heparin or dextran sulfate (see Recipes)

Equipment

1. Centrifuge with a swing bucket rotor (Thermo Fisher Scientific Heraeus Multifuge 3SR+ centrifuge )
   
2. Centrifuge Eppendorf 5702 with an A-4-38 swing bucket rotor (Thermo Fisher Scientific, catalog number: 05-400-318)
   
3. Vacuum pump (ILMVAC Biovac, modle: 104 )
   
4. 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)
   
5. Incubator at 37 °C in continuous gazing: 5% O₂, 5% CO₂ and 90% N₂ (Thermo Fisher Scientific Binder incubator, model: CB210 )
   
6. Laminar flow class II, type A2 biological safety cabinet (Fisher Scientific HeraSafe, catalog number: 13-998-002 )
   
7. VarioMACS^TM magnetic separator (Miltenyi Biotec)
   
8. MACS separation CS columns (Miltenyi Biotec, catalog number: 130-041-305 )
   
9. Chemistry Hood
   

Procedure