TITLE: Unconventional secretion of alpha-synucein mediated by palmitoylated DNAJC5 oligomers
AUTHORS: iona.thomas-wright, Richard Wade-Martins

[DESCRIPTION]
This protocol, carried out according to manufacturer’s instructions uses a classical ELISA experimental design with sensitive electrochemical detection to provide measure of 𝜶-synuclein concentration in the conditioned media of cultured iPSC-derived human cells.

[BEFORE_START]
Conditioned media used in this assay can come from any human cell line expressing 𝜶-synuclein though volumes and culture conditions have been optimised for iPSC-derived dopamine neurons differentiated according to the following protocol:
dx.doi.org/10.17504/protocols.io.q26g7y1jqgwz/v1 and plated at 100,000 cells per well in full area 96-well plates and cultured in 100 µL of media.

[GUIDELINES]
All aspects of the protocol for the MSD assay are carried out according to manufacturers instructions: summarised below. All reagents are included in the U-PLEX Human 𝜶-synuclein Kit unless otherwise stated.

[STEPS]
SECTION: Harvesting media and cell lysates
1. Harvest conditioned media from cells grown in a full-area 96 well plate, being careful not to disturb the cell monolayer. Add 30 uL RIPA lysis buffer (0.5% Triton X-100, 10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 0.5 mM EDTA,0.1% sodium dodecyl sulfate, 0.02% sodium deoxycholate, and (140 mM NaCl) to the cells remaining in each well and incubate on ice for 30 mins.
SECTION: Harvesting media and cell lysates
2. Centrifuge the harvested media 10 mins at 1000 xg to pellet floating cells. Collect the
supernatant to use in the assay.
SECTION: Harvesting media and cell lysates
3. Use a pipette to homogenise the cells in lysis buffer and then determine the total protein
concentration of the lysate using the Pierce BCA Protein Assay Kit according to manufactures
instructions.
SECTION: Measuring 𝜶-synuclein in conditioned media
4. Dilute the biotinylated capture anti-body 1:50 in Diluent 49 and add 25 µL to each well of the
provided plate. Seal the plate and incubate at room temperature for 1hr with agitation
SECTION: Measuring 𝜶-synuclein in conditioned media
5. During the incubation prepare the standard curve by serial dilution of the calibrator sample in
Diluent 49. For the highest concentration standard add 15 µL of calibrator to 285 µL to give a
10,000 pg/ml solution and then serially dilute 1:4 to give a range of standards between 2.44
pg/ml and 10,000 pg/ml. Use Diluent 49 alone as a blank.
SECTION: Measuring 𝜶-synuclein in conditioned media
6. Wash the plate 3 times to remove capture antibody with wash buffer (PBS containing 0.05%
Tween-20, prepare in advance)
SECTION: Measuring 𝜶-synuclein in conditioned media
7. Dilute detection antibody 1:50 in Diluent 49 and add 25 µL to each well.
SECTION: Measuring 𝜶-synuclein in conditioned media
8. Add 25 µL of conditioned media supernatant (prepared as described above) or 𝜶-synuclein
standard to each well. Seal the plate an incubate for 2 hrs at room temperature with agitation.
SECTION: Data Processing
11. Using the calibrator samples interpolate the 𝜶-synuclein concentration by fitting a polynomial in
the form y = ax2 + bx +c
SECTION: Measuring 𝜶-synuclein in conditioned media
9. Repeat step 3 to remove unbound sample and detection antibody 

SECTION: Measuring 𝜶-synuclein in conditioned media
10. Add 150 µL of Read Buffer and measure electrochemiluminescence on the MESO QuickPlex SQ
120 immediately
SECTION: Data Processing
12. Multiply the concentration of 𝜶-synuclein determined from the MSD assay by the total volume
of media in each well (100 μL) to obtain the total value in pico-grams released by the cells.
SECTION: Data Processing
13. Normalised release for difference in plating density/cell number by dividing by the total protein
values obtain by BCA