Materials and Reagents

1. Materials
   
   1. A 6-well cell culture plate (Corning, catalog number: 3506 )
   2. A 12-well cell culture plate (Corning, catalog number: 3512 )
   3. A 24-well cell culture plate (Corning, catalog number: 3524 )
   4. Pipette tips (Neptune Scientific, catalog numbers: BT20 , BT200 and BT1250 )
   5. A 1.5 ml tube with lid (INA•OPTIKA, catalog number: ST-0150F )
   
   
2. Cells
   
   1. RK13 cells: these cells were a gift from the Kitasato Institute
      Note: These cells are maintained in Eagle’s minimum essential medium (MEM) (see Recipes) containing 8% bovine serum (BS).
   2. Vero cells: our pre-existing stocks were originally obtained from American Type Culture Collection and have been maintained for more than 20 years in our laboratory
      Note: These cells are maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS), 100 unit/ml Penicillin, and 100 μg/ml Streptomycin.
   
   
3. Viruses
   
   1.  Rubella virus (RuV) TO-336 vaccine strain (Takeda Pharmaceutical) (Otsuki et al., 2011)
   2. RuV RVi/Hiroshima.JPN/01.03 wild type strain (Otsuki et al., 2011; Sakata et al., 2014)
   3. The recombinant RuV RVi/Hiroshima.JPN/01.03 strain expressing the green fluorescent AG1 protein (rHS/p150-AG1) (reported previously in Sakata et al., 2014)
   4. The recombinant measles virus (MeV) and human metapneumovirus (HMPV) expressing enhanced green fluorescent protein (EGFP) (MeV-IC323/Ed-H-EGFP and HMPV-rJPS02-76EGFP, respectively) (reported previously in Seki et al., 2006 and Shirogane et al., 2008)
      Notes:
      
      1. The TO-336 vaccine strain and RVi/Hiroshima.JPN/01.03 wild type strain were propagated in RK13 cells. The recombinant rHS/p150-AG1 was propagated in Vero cells. MeV and HMPV were propagated in Vero/hSLAM cells (Ono et al., 2001) and Vero/TMPRSS2 cells (Shirogane et al., 2008), respectively.
      2. Viruses were diluted with MEM before use. 
         
         
4. Virus-like particles of RuV (RuV-VLP)
   RuV-VLP, whose genome encodes Renilla luciferase and AG1 reporter genes, were produced and concentrated as described previously (Sakata et al., 2014), with some modifications (Otsuki et al., 2018).
   
   
5. Reagents
   
   1. Eagle’s MEM “Nissui” 1 (NISSUI PHARMACEUTICAL, catalog number: 05900 )
   2. Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D5796 )
   3. Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190144 )
   4. Fetal bovine serum (Biowest, catalog number: S1780-500 )
   5. Bovine serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16170078 )
   6. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
   7. L-Glutamine (200 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
   8. Agarose ME (IWAI CHEMICALS, catalog number: 50013R )
   9. 7.5% sodium bicarbonate (Thermo Fisher Scientific, Gibco^TM, catalog number: 25080094 )
   10. Sphingomyelinase from Bacillus cereus (Sigma-Aldrich, catalog number: S9396 )
   11. Neutral red solution (0.33%) (Sigma-Aldrich, catalog number: N2889 )
   12. Renilla Luciferase Assay system (Promega, catalog number: E2810 )
       Note: Lysis buffer and Renilla Luciferase Assay Reagent are included in this kit.
   13. MEM (see Recipes)
   14. 2x MEM (see Recipes)
   15. MEM containing 2% BS (or FBS) and 0.5% (or 0.4%) agarose (see Recipes)
   16. MEM containing 0.01% neutral red and 0.5% agarose (see Recipes)

Equipment

1. Micropipettes (Gilson, models: P10, P200, P1000, catalog numbers: F144802 , F123601 , F123602 )
2. Luminometer (Promega, model: GloMax^® 20/20 )
3. Autoclave (TOMY SEIKO, model: ES-215 )
4. Biosafety cabinet (Air Tech)
5. Cell culture incubator (at 35 °C and 37 °C with 5% CO₂) (Thermo Fisher Scientific, Forma, model: 310 )
6. Fluorescence microscope (ZEISS, model: Axio Observer D1 )
7. Cell culture microscope (Olympus, model: CKX53 )
   

Procedure