TITLE: Cytokine profiling analysis on conditioned medium of human neurons using Luminex multiplex assay
AUTHORS: Ching-Chieh Chou, Judith Frydman

[DESCRIPTION]
This protocol is used to identify secreted inflammatory factors by cytokine profiling of the conditioned medium from human transdifferentiated neurons of healthy donors and AD patients.

[STEPS]
SECTION: Sample collection
1. At post-induction day 35 to 38, human transdifferentiated neurons undergo the final round of medium change.
SECTION: Sample collection
2. Two days after medium change, a minimum of 200 µL of cell culture medium per
sample is collected to run duplicate wells without dilution.
SECTION: Sample collection
3. Conditioned medium is centrifuged at 10,000 X G for 10 min at room temperature to pellet out particulates.
SECTION: Sample collection
4. Supernatant is collected and immediately stored at −80°C.
SECTION: Sample collection
5. Cells are then trypsinized by 0.05% Trypsin-EDTA for 5 min and centrifuged at 200 X G for 5 min. Decant the supernatant and resuspend cells in a small aliquot of culture medium.
SECTION: Sample collection
6. Determine cell number for each sample by a hemocytometer for normalization of cytokine levels.
SECTION: Sample preparation and measurement
7. Thaw samples on ice prior to loading a 96-well filter plate for assay.
SECTION: Sample preparation and measurement
8. During this time, prepare Standards by rehydrating the lyophilized standard vial with Assay Buffer. Vortex and incubate on ice for 25 min. 
SECTION: Sample preparation and measurement
9. Perform serial dilutions in Assay Buffer and Standards must be used within 1 hr.
SECTION: Sample preparation and measurement
10. The 96-well filter plate is incubated with 120 µL/well of Reading Buffer for 10 min at room temperature on an orbital shaker at 500-600 rpm.
SECTION: Sample preparation and measurement
11. Completely remove Reading Buffer. Add 25 µL/well of samples or Standards and 25 µL/well of Assay Buffer.
SECTION: Sample preparation and measurement
12. Incubate with 50 µL/well of Antibody Beads for 2 hr at room temperature on an orbital shaker at 500-600 rpm. Then, transfer the plate to a refrigerator for overnight incubation at 4°C.
SECTION: Sample preparation and measurement
13. Next day, take out the plate from the refrigerator and stay at room temperature for 30 min without shaking.
SECTION: Sample preparation and measurement
14. Remove solutions and wash the plate with Wash Buffer for 3 times.
SECTION: Sample preparation and measurement
15. Add Detection Antibody with 25 µL/well and incubate for 2 hr at room temperature on an orbital shaker at 500-600 rpm.
SECTION: Sample preparation and measurement
16. Remove solutions and rinse the plate with Wash buffer for 3 times.
SECTION: Sample preparation and measurement
17. Add Streptavidin-PE with 50 µL/well and incubate for 40 min at room temperature on an orbital shaker at 500-600 rpm.
SECTION: Sample preparation and measurement
18. Remove solutions and wash the plate with Wash buffer for 3 times.
SECTION: Sample preparation and measurement
19. Add Reading Buffer with 120 µL/well and incubate for 5 min at room temperature on an orbital shaker at 500-600 rpm.
SECTION: Sample preparation and measurement
20. Read the plate on Luminex instruments following the manufacturer’s instructions.