Apr 07, 2026
CXCL1 supplementation on iPSC-Derived Midbrain Neurons
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. CXCL1 supplementation on iPSC-Derived Midbrain Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp7681gzp/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: April 07, 2026
Last Modified: April 07, 2026
Protocol Integer ID: 314537
Keywords: CXCL1, iPSC-derived neurons, midbrain neurons, conditioned media, astrocytes, primary midbrain neuronal culture, derived midbrain neuron, derived midbrain neurons this protocol, cxcl1 supplementation on ipsc, cxcl1 supplementation, concentrations of recombinant human cxcl1, recombinant human cxcl1, astrocyte, neuronal culture, induced pluripotent stem cell, primary midbrain, cell, media from atp13a2wt, atp13a2wt
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Abstract
This protocol aims to investigate the effects of varying concentrations of recombinant human CXCL1 on primary midbrain neuronal cultures derived from induced pluripotent stem cells (iPSCs) using conditioned media from ATP13A2WT astrocytes.
Materials
ATP13A2WT astrocyte conditioned media (Freshly prepared conditioned media) - 50 ml per treatment
Recombinant human CXCL1 (PeproTech, cat. no. 275-GR-010) - 1 vial (1 mg)
0.1% BSA in PBS (Vehicle/diluent) - 50 ml
Primary midbrain neuronal cultures (iPSC-derived, plated on day 25 of differentiation) - 1 culture dish
Reagents:
Protein Recombinant CXCL1 - Chemokine involved in immune response and neuronal signaling
Buffer 0.1% BSA in PBS - Bovine serum albumin used as a stabilizer and diluent
Troubleshooting
Problem
Neurons not adhering to the plate
Solution
Ensure proper coating of plates and check cell viability before plating.
Problem
Inconsistent CXCL1 concentrations during media changes
Solution
Prepare fresh media and verify dilutions before each change.
Safety warnings
Handle all reagents and materials according to safety data sheets. Use personal protective equipment (gloves, lab coat, goggles) when handling biological materials and reagents.
Cell Plating
On day 25 of differentiation, plate primary midbrain neurons under standard culture conditions.
Add 100,000 neurons per well off a 96w plate (100µl). Incubate at 37 °C, 5% CO2 for 24 hours.
CXCL1 Preparation and Treatment
Prepare recombinant CXCL1 stock and initiate treatment. Reconstitute CXCL1 in 0.1% BSA in PBS to a final concentration of 1 mg/ml. Dilute to working concentrations (1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml) in astrocyte conditioned media.
Media Change and Treatment Initiation
On day 26, perform the first half media change with supplemented CM or vehicle-only CM for controls. Remove 50% of the existing media volume (50 µl) and replace with an equal volume (250 µl) of freshly prepared supplemented CM. For controls, use 0.1% BSA in PBS.
Ongoing Treatment
Continue half media changes every other day for four weeks.
Downstream Analyses
At the end of the four-week exposure period, proceed to downstream analyses.
Protocol references