Aug 30, 2024
Nuclei counting after Trypan Blue staining
- 1Karolinska Institutet, Department of Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology, Stockholm 171 77, Sweden
Protocol Citation: Carmen Abaurre, Ka Wai Lee, Ernest Arenas 2024. Nuclei counting after Trypan Blue staining. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82838l2w/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: August 28, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 106552
Keywords: nuclei isolation
Abstract
Nuclei counting after Trypan Blue staining
Guidelines
Be wary of nuclei integrity when counting for snRNA-seq. Some minor blebbing of nuclei can still be considered acceptable, but broken up nuclei are considered debris and should not be counted, as they have lost single-cell characteristics.
Trypan Blue also stains debris, so for counting it is important to have a clean nuclei suspension.
Materials
Nuclei suspension
Hemocytometer
Trypan Blue 0.4%
Microscope
Tally counter
0.2 ml tube
Prepare nuclei suspension from samples.
In order to increase accuracy, prepare duplicates or triplicates per sample.
Add 9 μl of 0.4% Trypan Blue to a 0.2 ml tube
Add 1 µl of nuclei suspension to the Trypan Blue solution. Do not dispense to the walls of the tube.
Mix slowly by pipetting up and down 10 times. If the nuclei preparation is prone to clumping or losing integrity (breakage and release of chromatin), use a 20 µL pipette and further reduce mixing speed.
Fill the counting chamber. Observe at 20X
Calculate the average concentration obtained across duplicates/triplicates of the sample