We recommend that cells are fixed prior to isolation in order to preserve their transcriptomic state . Importantly, dead cells will not be compatible with LUTHOR HD library preparationand 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.
Cell fixation guidelines have been validated internally and are outlined below:
...
Buffer | Stock | Final concentration | To be added per Sample (μl) |
Fixation Buffer (FB) | 37% PFA (F1635) 1x PBS pH7.4 (10010-023) | 4% # | 108.1 891.9 |
Fixation/Permeabilization Buffer (FPB) | 37% PFA (F1635) 1x PBS pH7.4 (10010-023) 10% Triton X-100 | 4% # 0.1% | 108.1 881.9 10 |
Quenching Buffer (QB) | 1M Tris-HCl pH8 1x PBS pH7.4 (10010-023) 40 U/μl RNase Inhibitor (Y9240L) | 200 mM # 0.16 U/ μl | 200 798 2 |
Resuspension Buffer (RB) | MB-H20, molecular biology grade 1x PBS pH7.4 (10010-023) 40 U/μl RNase Inhibitor (Y9240L) | # # 0.16 U/ μl | 500 498 2 |
Specific Reagents
Vendor | ItemLot | Cat. No. |
Sigma-Aldrich | DMSO (99.5 %) | D4540ThermoFisher |
Thermo Fisher Scientific | Glycerol* (99%, MBG) | J61059 |
Thermo Fisher Scientific | PBS, pH 7.4 (no Ca, no Mg, no Phenol Red) | 10010-023 |
Sigma-Aldrich | PFA (formaldehyde solution) | F1635 |
Qiagen | RNase inhibitor, 40 U/μl | Y9240L |
*Mix 1:1 nuclease-free water with 99% Glycerol, filter through a 0.2 μm filter and store at room temperature in LoBind tubes
...
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 a 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.
...
12. Pass cells through 40 μm strainer.
13. Count and determine viability of 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% non viable cells. We recommend staining with fluorescent dyes such as Acridine Orange and Propidium Iodide (AO/PI). AO stains all cells, while PI only stains dead and fixed cells. Inefficient fixation will result into lower percentages of PI-stained cells (i.e., higher percentage of non-fluorescent cells).
Fixed Sample Storage Guidelines
...