We recommend that cells are fixed prior to isolation in order to preserve their transcriptomic state and recommend to work with cells showing a viability of at least 90%.
Recommendations to assess viability:
a. Trypan Blue staining
b. Dual Acridine Orange (AO)/Propidium Iodide (PI) staining. AO will stain live cells in green, while PI will stain dead cells in red.
NOTE: Trypan Blue stains debris, and if these are of similar size as cells, they can be wrongly counted as cells.
Therefore, when many debris are present, fluorescent staining AO/PI is preferred, since it will only stain cells.
...
Pass cells through 40 μm strainer.
Centrifuge at 500 g for 3 min at 4-8ᵒC.
Remove the supernatant without disturbing the pellet.
Add 1 ml of freshly prepared Fixation Buffer (FB) or Fixation/Permeabilization Buffer (FPB).
Note: always prepare fresh FB
Gently pipette-mix 5x.
Incubate for 12-24 h at 4ᵒC (for storage or shipment), or 1 h at 20ᵒC for immediate use.
Centrifuge at 500g for 3 min at 4-8ᵒC.
Remove the supernatant without disturbing the pellet.
Add 1 ml cold Quenching Buffer (QB) and gently resuspend cells. Keep on ice for 5 min.
See storage guidelines.10.
Centrifuge at 500 g for 3 min at 4-8ᵒC.
...
Resuspend cells in 1 ml Resuspension Buffer (RB).
...
Pass cells through 40 μm strainer.
...
Count cells. Even when cells die after fixation, membrane may not be compromised, and may therefore prevent PI from entering the cell. Our recommendation is therefore to fix and permeabilize cells, ensuring PI staining of all dead cells. An efficient fixation protocol should give >95% dead cells. Inefficient fixation will result into lower percentages of PI-stained cells (i.e., higher percentage of non-fluorescent cells).
Add 2 µl of fixed cells (previously resuspended in RB, step 11) to 3 µl of LUTHOR HD Cell Lysis Buffer (CLB), then proceed with the LUTHOR HD protocol as outlined in the User Guide (Step 3, page 13).
Fixed Sample Storage Guidelines
...