Apr 09, 2026
iGLUT and iGABA dissociation
- Meilin Fernandez Garcia1,
- Kristen Brennand1
- 1Yale University School of Medicine
- Brennand Laboratory
- Brennand Lab
Protocol Citation: Meilin Fernandez Garcia, Kristen Brennand 2026. iGLUT and iGABA dissociation. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4rj5lx1/v1
Manuscript citation:
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 08, 2026
Last Modified: April 09, 2026
Protocol Integer ID: 314674
Keywords: CRISPR, stem cells, neurogenomics, igaba dissociation dissociation of npc, igaba dissociation dissociation, igabas to viable single cell, igaba, viable single cell, iglut, single cell, cell
Funders Acknowledgements:
NIMH
Grant ID: RM1MH132648
Abstract
Dissociation of NPCs, immature and mature iGLUTs and iGABAs to viable single cells.
Guidelines
- Set up at least twice (1 week separated from each other). You will appreciate the backup.
- Don’t forget to add DNAse; make sure papain is not expired.
- Incubating longer than 15 min decreases cell viability and doesn't improve singleness of cells.
- If you are working with a lot of plates, you can stop the enzyme action after 10 mins papain incubation, and manually break down the cells afterward.
- Incubate at 39C NOT 37C! It increases efficiency of dissociation and use a shaker in slow speed 125 rpm.
- DO NOT WASH CELLS IN NEURON MEDIA. Use DMEM+FBS to stop enzymatic dissociation
- Spin cells at low speed 600g NOT 1 krcf. They will clump otherwise.
- Before straining, wait a bit for cell debris to settle, take the cells, and count that.
- Don't overdilute your cells: 10x will fail if too diluted.
Materials
Papain Suspension 42.0 mg/mL (Worthington cat no. LS003127)
Hank’s balanced salt solution (HBSS) with added HEPES
DMEM (ThermoFisher Scientific, #10566-016)
FBS (Gibco 10438-026) 0.5M
EDTA
Option 1: Papain Dissociation
Aspire medium
Wash wells with PBS-EDTA 0.5mM (e.g., 0.5 mL per well of a 12-well-plate; recline the cells from tube rack to disturb the neurons as little as possible). Aspirate.
Add 1/4 of well volume of papain-HBSS-EDTA-HEPES solution (e.g., 250 µL per well of a 12-well-plate or 125 µL per well of a 24-well-plate)
Incubate at 37°C for 10-15 min in shaker at 125 rpm (bacterial shaker taped or inside metal holders). Tap cells on the side every 5 min.
Pipette cell clumps up and down to break cells.
Transfer dissociated cells to a 15 mL tube plate with 6 mL of DMEM-10%FBS (2 volume dilution of papain) and wash wells with 5 mL (e.g., 400 µL /well of a 12-well-plate)
3 mL cells suspension+ 6 mL Medium+ 5 mL Medium wash for each plate.
NOTE: There should almost no cell clumps.
Spin at 600g, 5 min, RT. Decant medium by inverting the tube.
Resuspend in 1-2 mL of DMEM-10%FBS/plate.
Pass cells through strainer on top of a 15 mL tube.
Count cells in hiPSCs setting
Option 2: Accutase Dissociation
Wash w. PBS ((-Ca/-Mg) twice
0.5 mL Accutase, w/Rock inhibitor, 37°C, ~25 min, frequent agitation
Pipet gently w. p1000,
Wash medium w/FBS, and add 15 mL conical tube,
Spin, 300 g, 5min, 4°C, aspirate sup,
Re-suspended in neuron media w. 10% FBS and Ri.
Hashing (if necessary)
Spin, 300 g, 5min, RT, remove sup
Add 100 µL staining buffer (2%BSA/0.02% tween20, RNAase inhibitor)
Add 10 µL FcX. resuspend, 10min, 4C (Fc block)
Add 2 µL (1µg) hashing Ab for each sample, 30min, @4C
Wash w.1 mL staining buffer, spin, 300 g, 5 min,
Repeat washing 3 times.
Cell Counting
Counting: 2.5 µL cell suspension + 2.5 µL staining buffer (1:2 dilution) + 5 µL Trypan
blue
Ensure >60% viability