Feb 18, 2025
ICC Staining of Fixed Organoid Cells for Flow Analysis
- 1Wyss Institute
Protocol Citation: Katharina Meyer 2025. ICC Staining of Fixed Organoid Cells for Flow Analysis . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6de6dvqe/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: February 18, 2025
Last Modified: February 18, 2025
Protocol Integer ID: 120848
Abstract
Indirect ICC Staining of PFA Fixed Dissociated Organoid Cells for Flow Analysis
Dissociation of Organoids
Dissociation of Organoids
Wash organoids in each well with PBS 2 times.
Transfer organoids into a 2-ml Eppendorf tube containing 300-500 µl of cold Cell Recovery Solution for Matrigel embedded organoids (Corning).
Incubate on ice for 1 hour.
Spin down at 1000g for 1 minute.
Remove supernatant and wash with 1 ml cold PBS once.
Incubate organoids with 300-500 µl cold Accutase for 15 minutes.
Spin down at 1000g for 1 minute.
Remove supernatant and wash with cold PBS once.
Dissociate and resuspend the cells in 500 µl cold 4% PFA to start fixation.
Incubate on ice for 1 hour.
Spin down at 3000g for 1 minute.
Remove supernatant and wash with 3 x 1 ml PBS. Resuspend pellet each time by vortexing.
Keep cells in PBS until permeabilization.
ICC Staining
ICC Staining
Recover cells by spinning down at 3000g for 1 minute.
Count cells.
Remove supernatant and resuspend the pellet with 300 µl blocking buffer (3% Normal donkey serum and 0.3% Triton X-100 in PBS).
Incubate for 30 minutes and briefly vortex the cells at 30% speed.
Spin down at 3000g for 1 minute.
Remove supernatant and resuspend the cells with blocking buffer + primary antibody/ies (100-250 µl and minimum of 100K cells) via vortexing at 70% speed.
Lay all tubes on a rack holder and secure them with tape.
Incubate on a shaker at 4°C overnight.
Wash the cells 3 x 500 µl PBS. Spin down at 3000g for 1 minute.
Resuspend the cells with 100-250 µl of blocking buffer + secondary antibody/ies.
Incubate the cells at room temperature for 1 hour and briefly vortex the cells at 30% speed.
Wash the cells 3 x 500 µl PBS. Spin down at 3000g for 1 minute.
Resuspend the cells in 0.5-1 ml PBS and filter the cells through a mesh of 0.45 µm. Keep the cells from light. On ice.