Nov 18, 2025
Protocols Suite — SH-SY5Y / iCell® Neurons / High-Content Imaging
- Sara Lucas1,
- Giuseppe Uras1,
- Sofia Koletsi2,
- federico fierli2,
- veronica lentini2,
- david chau2,
- anthony schapira2
- 1UCL;
- 2University College London, University of London
Protocol Citation: Sara Lucas, Giuseppe Uras, Sofia Koletsi, federico fierli, veronica lentini, david chau, anthony schapira 2025. Protocols Suite — SH-SY5Y / iCell® Neurons / High-Content Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95zozl3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 12, 2025
Last Modified: November 18, 2025
Protocol Integer ID: 229622
Keywords: Cell culture, Neuronal differentiation, Compound treatments, Cell staining, High-content imaging, sy5y cell, using retinoic acid, like cells for downstream assay, retinoic acid, neuron, glutamax, sy5y, dapi nuclear stain
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP000420
Abstract
Routine maintenance of SH-SY5Y cells and SH-SY5Y lines overexpressing 1K/2K/3K-SNCA in DMEM/F-12 with GlutaMAX, 10% FBS, and 1× Pen/Strep.
Differentiation of SH-SY5Y (WT or 3K-SNCA) using retinoic acid (RA) plus BDNF to generate neuron-like cells for downstream assays.
Culture of iCell® DopaNeurons with heterozygous SNCA p.A53T (Fujifilm CDI R1109) and isogenic control (R1088) using vendor media; optional 7-day compound treatment with half-medium changes.
Short-term (24 h) and extended (7-day) treatments in SH-SY5Y and iCell® neurons.
Fixation, permeabilisation, and immunostaining for α-syn, pSer129 α-syn, TFEB, GBA, LAMP1; DAPI nuclear stain; acquisition on Opera Phenix® Plus; analysis in Harmony/Columbus.
Materials
- SH-SY5Y (ATCC CRL-2266; RRID:CVCL_0019)
- DMEM/F-12, GlutaMAX™ (Gibco 31331-028)
- FBS, qualified (Gibco 10270-106)
- Penicillin–Streptomycin (10,000 U/mL) (Gibco 15140-122)
- Trypsin 2.5% (Gibco 15090-046)
- Versene® (EDTA) 1× (Gibco 15040-066)
- PBS, sterile (1×)
- Tissue culture plastics (T-25/T-75 flasks; 6-/12-/96-well plates)
- CO₂ incubator (37 °C, 5% CO₂), Class II hood, centrifuge, inverted microscope
- Neurobasal™ Medium (Gibco 21103-049)
- B-27™ Supplement (50×) (Gibco 17504-044)
- GlutaMAX™ Supplement (Gibco 35050-061)
- Retinoic acid (RA) (prepare 30 µM working)
- Recombinant human BDNF (Cell Signaling 3897 / 3897S) — use 5 ng/mL
- Geltrex® LDEV-free (Gibco A1413301 / A1413302)
- iCell® DopaNeurons PD SNCA A53T HZ (FCDI R1109)
- iCell® DopaNeurons (isogenic control) (FCDI R1088)
- iCell® Neural Medium (FCDI M0100)
- iCell® Neural Supplement B (FCDI M1029)
- iCell® Nervous System Supplement (FCDI M1031)
- Vendor-recommended coating (per current datasheet)
- YM201636 (Cayman Chemical 13576) — make 10 mM stock in DMSO
- Ambroxol hydrochloride (Merck/Sigma A9797) — make 10 mM stock in DMSO
- Sterile DMSO (vehicle), sterile PBS
- SH-SY5Y or differentiated SH-SY5Y; iCell® neurons
- 4% Paraformaldehyde (PFA) in PBS (Severn Biotech 40-7401-05)
- Methanol, ice-cold (100%)
- Normal Goat Serum (NGS) 5% (Abcam ab7481)
- α-Synuclein, rabbit mAb (Abcam ab138501; RRID:AB_2537217) (1:750)
- GBA, mouse mAb clone 2E2 (Millipore AP1140-100UG; RRID:AB_11211799) (1:1000)
- TFEB, rabbit mAb D2O7D (Cell Signaling #37785) (1:500)
- LAMP1, mouse mAb clone 25 (BD 611042; RRID:AB_398355) (1:1000)
- α-Synuclein, mouse mAb clone 4D6 (Abcam ab1903; RRID:AB_302665) (1:750)
- Phospho-Ser129 α-syn, mouse mAb (FUJIFILM Wako 015-25191; RRID:AB_2572224) (1:1000)
- Goat anti-mouse Alexa Fluor 488 (A-11017; RRID:AB_2534084) (1:2000)
- Goat anti-rabbit Alexa Fluor 568 (A-11011; RRID:AB_143157) (1:2000)
- Goat anti-rabbit Alexa Fluor 647 (A-21244; RRID:AB_2535812) (1:2000)
- DAPI (Thermo D1306) — 1 µg/mL, 10 min
- Opera Phenix® Plus High-Content Screening System (RRID:SCR_021100)
- Harmony® High-Content Imaging Analysis Software (RRID per site)
- Columbus® Image Data Storage Analysis (web-based)
Troubleshooting
Safety warnings
Follow BSL-2 aseptic technique; wear appropriate PPE.
RA is light-sensitive and teratogenic; handle under low light and use PPE.
DMSO facilitates skin absorption; YM201636 perturbs endolysosomal trafficking. Use PPE.
PFA and methanol are hazardous; use a fume hood and appropriate PPE.
Before start
- Thaw FBS and supplements at 4 °C; warm media to room temperature.
- Coating: Dilute Geltrex 1:100 in cold DMEM/F-12 on ice; add to plates and incubate 1 h RT (or overnight 4 °C). Aspirate immediately before seeding.
- Review the latest FCDI handling guide for substrate and thawing specifics.
Procedure — Staining
Remove medium; rinse once with PBS.
Fix with 100 µL/well 4% PFA, 20 min RT.
Permeabilise with 100 µL ice-cold methanol, 20 min.
Wash with PBS 3×.
Block with 5% NGS in PBS, 1 h RT.
Primary antibody in blocking buffer; incubate overnight at 4 °C.
Wash PBS 3×.
Secondary antibody (1:2000 in block), 2 h RT, protect from light.
Wash PBS 3×.
DAPI 1 µg/mL, 10 min → wash PBS 3× → leave 200 µL PBS/well. Store 4 °C, dark, until imaging.
Procedure — High-Content Imaging (Opera Phenix® Plus)
Objective: 63×; Mode: Confocal.
Fields: Acquire 5–10 random fields/well.
Channels (separate acquisitions): DAPI ~50 ms/~30% laser; AF488 ~100 ms/~70%; AF568 ~100 ms/~70%; AF647 ~100 ms/~80%.
Z-stack: 2 µm step; compute Maximum-Intensity Projection (MIP).
Save raw images with plate, well, field, channel, exposure metadata.
Procedure — Image Analysis (Columbus/Harmony)
Import MIP z-stacks (if not already done in acquisition).
Segment nuclei on DAPI (Columbus Method B).
Define cytoplasm expanding from nuclei; use α-syn (488) to assist (Columbus Method A).
α-Syn inclusions (488): Cytoplasmic mask; intensity filter 5,500–20,000 a.u.; extract # inclusions/field and MFI of foci; normalise by nuclei count → inclusions/cell.
TFEB partitioning (568): Nuclear vs cytoplasmic ROIs; intensity range 150–20,000 a.u.; compute MFI/cell for nuclei and cytoplasm (normalise by nuclei).
GBA (568): Cytoplasmic intensity range 600–20,000 a.u.; mean cytoplasmic intensity/cell.
LAMP1 (568): Cytoplasmic mask; 600–20,000 a.u.; raw sum intensity/cell (or puncta counts if spot detection enabled).
pSer129 α-syn (488): Cytoplasmic mask; 1,000–1,800 a.u.; # inclusions/cell.
QC Export: Exclude fields with 50 cells or saturated pixels; export per-field, per-well summaries for statistics.
Expected results
Robust nuclear DAPI; discrete α-syn cytoplasmic inclusions when present; measurable TFEB nuclear/cytoplasmic shift; LAMP1 puncta in perinuclear region.
Troubleshooting
Weak signal — verify antibody dilutions and fixation/permeabilisation consistency; adjust exposure to avoid saturation.