Fig. 1
Illustration of full-length MOG fused to EGFP as displayed by the transfected cells. The cartoon of MOG is based on Fig. 1b from (18)
Fig. 2
Quantitative analysis of the anti-MOG response. FACS plots of HeLa cells transfected with EGFP alone (left) or with MOG-EGFP (right) that were incubated with a serum sample which scored positive for anti-MOG (FACS ratio 10.7). To determine the MOG reactivity, we gate on the cells inside the dashed rectangle and calculate the FACS ratio as MFI (MOG-EGFP)/MFI (EGFP only)
2 Materials
1.
Cell culture facility: For the culture of HeLa cells we use Dulbecco’s modified Eagle’s medium supplied with 10 % fetal calf serum (FCS) and 1 % penicillin-streptomycin (Pen Strep).
2.
Refrigerated centrifuge with swing rotor for 96-well plates.
3.
Phosphate buffer saline (PBS), trypsin/EDTA, FACS buffer (1 % FCS in PBS).
4.
Transfection reagent (Lipofectamine 2000®, Invitrogen, Carlsbad, California, USA).
5.
6.
Murine anti-MOG monoclonal antibody (mAb) (e.g., 8-18C5) as control of surface expression of MOG in the transfectants and F(ab’)2 Biotin-SP goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) (see Note 2 ).
7.
Human serum samples to test for autoantibodies to MOG.
8.
F(ab’)2 Biotin-SP-conjugated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA).
9.
Propidium iodide for dead cell staining.
10.
AlexaFluor® 647-conjugated Streptavidin (Jackson ImmunoResearch, West Grove, PA, USA).
11.
Flow cytometer (e.g., BD Bioscience FacsVerse™, San Jose, CA, USA).
3 Methods
1.
Keep cells growing in culture medium and collect them for the experiment when they are 80 % confluent.
2.
Wash cells once with PBS and remove them from plate with trypsin/EDTA.
3.
Suspend detached cells in culture medium and collect them in a 15 ml plastic tube.
4.
Centrifuge for 5 min at 200 × g and suspend in 5 ml culture medium.
5.
Seed cells at 1.5 × 105/ml in culture medium (see Note 3 ) and divide them into two cell culture dishes (for a 10 cm culture dish we use 10 ml of cell suspension).
6.
Transfect cells in one dish with MOG-EGFP and cells in the other dish with EGFP when they are 90 % confluent.
7.
Collect cells 24 h after transfection; remove them from plate as described in steps 2–4.
8.
For determination of anti-MOG serum abs, transfer cells to a 96-well plate for flow cytometry. Use 50,000 cells/well.
9.
Centrifuge for 5 min at 200 × g and wash cells by adding to the pellet 150 μl of FACS buffer. Repeat this step once more (see Note 4 ).
10.
Incubate cells with a 1:50 human serum dilution in FACS buffer for 45 min at 4 °C. Cells used as a control for surface expression of the MOG construct are incubated with the mAb 8-18C5 (0.5 μg/ml).
11.
Wash cells three times with FACS buffer as previously described and incubate them with a 1:500 dilution of the biotin-SP-conjugated goat anti-human or anti-mouse IgG for 30 min at 4 °C (see Note 5 ).
12.
Wash cells as above and incubate them in the dark with a 1:2,000 dilution of AlexaFluor® 647-conjugated streptavidin for 30 min at 4 °C.
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