Appendix e-1: Method for aquaporin 4 antibody detection
Briefly HEK 293 cells were plated on coverslip coated with poly-L-lysine and inserted in 24 wells plates to allow cell growth. Twenty-four hours later, cells were transfected using transfection reagent Lipofectamine LTX (Life Technologies, NY). Twenty-four hours after transfection, cells were washed with cold phosphate buffer, then fixed using 4% paraformaldehyde for 10 minutes at room temperature and rinsed three times in phosphate buffer (fixed cells protocol). Cells were incubated with patient or control serum samples (1:20 dilution) in phosphate buffer plus 5% normal goat serum and 0,05% Triton for 90 minutes at room temperature. They were rinsed three times in phosphate buffer and incubated for the detection of IgG antibodies with goat anti-human IgG Cy3 conjugated secondary antibody at 1: 20 000 dilution (Bioscience Research Reagents, CA) for 90 minutes at room temperature. Nuclei were counterstained by diamidino-2-phenylindole (DAPI, 1:100 dilution, 0,09 µg/ml). Coverslip were then mounted on slides with Mowiol solution. Slides were stored, light protected at 4°C and then imaged on a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany). Slides were coded and scored for the intensity of immunofluorescence. Three independent assessors scored sera negative or positive unaware of the clinical diagnosis (RBV, GC, RM). No titration was performed. No serum was classified indeterminate or equivocal. Alternatively, for live cell protocol, cells were washed and incubated with Dubelcco’s Modified Eagle Medium (DMEM) with 1% bovine serum albumine all along the experiments. The contact between cells and patients’ sera diluted in DMEM- bovine serum albumine (1:20) was made during 30 minutes at room temperature. Cells were then fixed after sera incubation10 minutes at room temperature with 1% paraformaldehyde in DMEM, which was termed “post-fixation”, and then incubated for the detection of IgG antibodies with goat anti-human IgG Cy3 conjugated secondary antibody (1: 20 000 dilution).
For the purpose of this comparison approach we used three different plasmids to be expressed in HEK cells: 1- the full length Human AQP4-M1 isoform cDNA was obtained from Origene (OriGene Technologies Inc., Rockville, MD) and cloned into plasmid enhanced green fluorescent protein -C1 (Clontech, Saint-Germain-en-Laye, France) using the reverse primer 5’-CCGGTACCTCATACTGAAGACAATACC introducing a Kpn1 site at the 3’ end and the forward primer 5’-GAAGATCTATGAGTGACAGACCCAC introducing a Bgl2 site at the 5’ end. The PCR product was inserted into the Bgl2 and Kpn1 sites of the enhanced green fluorescent protein vector and the DNA sequence was verified by sequencing; 2- the truncated Human AQP4-M23 isoform plasmids tagged N-terminally with enhanced green fluorescent protein, or untagged, were kindly provided by P. Waters (Neuroimmunology Group, Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom); 3- enhanced green fluorescent protein-C1 plasmid (Clontech, Saint-Germain-en-Laye, France) was also used in cotransfection with untagged AQP4.