Sep 21, 2022
Flow cytometry
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. Flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3956eg25/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: September 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70344
Keywords: ASAPCRN
Abstract
This protocol is for immunolabelling fixed midbrain dopaminergic neurons for flow cytometry analysis and downstream flow cytometry acquisition.
Sample preparation
Sample preparation
Cells were washed once with PBS
The cells were incubated with Accutase (Gibco) to generate a single-cell suspension for 00:05:00 at 37 °C
5m
A cell suspension of 500k/ml was prepared in media
Cells were then spun down at 200 x g, 00:05:00 , and the supernatant was removed.
5m
Cell pellet was resuspended gently in 4ml of 4% paraformaldehyde and briefly vortexed at a low speed before being spun on a rotation spinner for 00:10:00 at room temperature.
10m
After fixation, samples were spun down and supernatant removed
Cells were resuspended in 2ml of 0.1% BSA (Sigma) in PBS.
After resuspension, cells were filtered through a 70μm strainer (Miltenyi Biotec) to filter out any cell clumps
Cells were then centrifuged 200 x g, 00:05:00 , and the supernatant was removed.
5m
Cell pellets were then resuspended in 1ml of permeabilization/blocking buffer (0.1% Triton X-100, 1% BSA, 10% normal goat serum (Sigma) in PBS), and incubated on a rotation spinner for 00:30:00 at room temperature.
30m
After permeabilization/blocking, cells were centrifuged 200 x g, 00:05:00 and the supernatant was removed.
5m
Cells were then resuspended in the primary antibodies (1:200) made up in 0.1% BSA in PBS, and incubated on the rotation spinner for 01:00:00 at room temperature.
1h
After primary antibody incubation, cells were centrifuged 200 x g, 00:05:00 , supernatant removed and washed once in 0.1% BSA in PBS.
5m
Cells were then resuspended in the species-specific secondary antibodies (AlexaFluor 488, 647) at a dilution of 1:500 made up in 0.1% BSA in PBS and incubated in the dark on a rotation spinner for 00:30:00 .
30m
After incubation, cells were centrifuged 200 x g, 00:05:00 , supernatant removed and washed once in PBS, followed by incubation with DAPI made up in PBS for 00:05:00 .
10m
The DAPI + PBS was then removed, followed by one wash in PBS, before being analysed on the flow cytometer.
Cell sorting and analysis
Cell sorting and analysis
The samples were run on the LSRii (BD) cell sorter. Scattering was initially used to discard debris as well as cell doublets and larger clumps.
The single-cell population was then gated to include DAPI positive only cells (negative control).
The gating threshold for measured channels was determined using the control lacking the antibody of interest (Fluorescence minus one (FMO) control), for both channels being recorded.
Once the parameters had been set, 10,000 cell events were recorded, and data were processed and analysed on the FlowJo software.