Jun 30, 2025
TH-TdTomato expression measurement by FACS
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2025. TH-TdTomato expression measurement by FACS. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjbk7xgk5/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: June 18, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 220539
Keywords: ASAPCRN, tdtomato expression measurement by fac, tdtomato expression measurement, analysis of midbrain neuron, midbrain neuron, cytometry, td, neuron
Abstract
This protocol outlines the preparation and flow cytometry-based analysis of midbrain neurons differentiated from Td-Tomato reporter hPSCs.
Troubleshooting
Cell Culture and Preparation
Differentiate TH-TdTomato lines into midbrain neurons in 6-well plates until the desired differentiation day.
Confirm cell morphology and fluorescence (if applicable) before harvesting.
Cell Detachment
Remove media and wash cells once with PBS
Gently lift neurons using Accutase supplemented with ROCK inhibitor (Y-27632). 1ml per well of a 6w plate
Incubate as needed (typically ~5 minutes) at RT until cells detach.
Collect cells into tubes with twice the volume of culture medium containing ROCK inhibitor.
Centrifuge the cell suspension at 300 × g for 5 minutes at room temperature.
Carefully remove the supernatant.
Resuspend the pellet in FACS buffer: PBS + 1x B-27 supplement + ROCK inhibitor (Y-27632) + DAPI (final concentration 1µg/ml) for live/dead discrimination
Keep cells on ice from this step forward
Make sure to include a sample with no DAPI for proper gating
For gating: collect TdTomato-negative line, or undifferentiated iPSC as a negative control to establish the TdTomato gate.
Pass the resuspended cells through a 35 µm cell strainer into FACS-compatible tubes
Flow citometry (FACS)
Analyze TdTomato fluorescence using a flow cytometer with appropriate excitation/emission settings (e.g., 554/581 nm) and DAPI (Ex/Em ~358/461 nm).
Record at least 10,000 events from live population
Gating Strategy:
FSC/SSC: Gate to exclude debris and cell clumps.
DAPI-negative cells: Gate for live cells excluding DAPI+ dead cells
TdTomato fluorescence:
Set gate using Td-tomato negative line
Identify TdTomato+ neurons and MFI from live population.
Analysis with proper software (FlowJo)