- [Instructor] Flow cytometry
is a popular laser-based technology
to analyse the characteristics of cells or particles.
This video will teach you
how to prepare and stain your cells
for flow cytometry analysis.
What you will need is a bio-hazardous waste container,
wash buffer, DAPI, blocking buffer,
cells, 96 well plate, primary and secondary antibodies
or conjugated primary antibody,
pipettes and tips, Eppendorf tubes,
waste for biological liquid, a centrifuge and timer.
If you are using an antibody for the first time,
the working concentration
should be determined via titration.
Titration is important to avoid excess antibody
which can result in the antibody binding
to low affinity targets.
This low affinity binding
can increase background fluorescence.
Use your antibody's ideal working concentration
and dilute the primary antibody in wash buffer.
After washing your cells,
re-suspend them at approximately 1-10 million cells per mL
in ice-cold wash buffer with sodium azide.
Add 100 μL of the cell suspension
to a 96-well plate with a U-shaped bottom.
Cell viability should be measured using a marker
for live and dead cells.
Viability should be around 95% and not less than 90%.
Then centrifuge the cells at 200g for five minutes
and discard the supernatant.
Re-suspend your cells in the blocking buffer
which consists of PBS with 10% serum.
However, the type of serum will depend on the host-species
of your primary and secondary antibodies.
Block the cells for 30 minutes at four degrees celsius.
Centrifuge the cells at 200g for five minutes.
Make sure the pellet is visible in the plate.
Then discard the supernatant.
Add the diluted antibody solutions from the first step
to the corresponding wells.
To keep the unstained control well free from antibodies,
add wash buffer instead of the antibody solution
to this well.
Re-suspend all the wells to ensure all the cells
are in contact with the antibody solution.
Incubate for 30 minutes at four degrees celsius in the dark.
Wash the cells three times by centrifugation
at 200g for five minutes
and re-suspend them in ice-cold wash buffer.
You may need to adjust the conditions of the centrifugation,
force and time for the cell types used.
If you are using a conjugated primary antibody,
skip to the addition of the live dead staining.
Dilute the secondary antibody, in this case,
a goat to mouse with an A488 fluorochrome
in wash buffer at the optimal dilution determined
by the user according to the manufacturer's instructions
and then suspend the cells in this solution.
Incubate for 30 minutes at four degrees celsius.
This incubation must be done in the dark.
Wash the cells three times by centrifugation
at 200g for five minutes
and re-suspend them in ice-cold wash buffer.
These washes remove excess secondary antibody
to reduce background fluorescence.
As before, ensure the pellet is visible in the plate
before discarding the supernatant.
Check for live/dead cells by staining with DAPI
or other viability dyes
for five minutes at four degrees in the dark
before analysis at the flow cytometer.
Or if your cells are being subsequently fixed,
include a fixable live/dead cell marker.
Store the cell suspension immediately
at four degree celsius in the dark.
For best results, analyse the cells on the flow cytometer
as soon as possible.
We recommend analysis on the same day
or as soon as possible.
For extended storage, as well as for greater flexibility
in planning time on the cytometer,
fix the cells with 1-4% paraformaldehyde
to prevent deterioration.
Ensure you wash a couple of times
before storing appropriate buffer.
For this, make sure you use a fixable live/dead dye.