Can cells be fixed and then processed with LUTHOR HD?
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.
Cell fixation guidelines have been validated internally and are outlined below:
Buffers
Buffer | Stock | Final concentration | To be added per Sample (μl) |
Fixation Buffer (FB) | 37% PFA 1x PBS pH7.4 | 4% # | 108.1 891.9 |
Fixation/Permeabilization Buffer (FPB) | 37% PFA 1x PBS pH7.4 10% Triton X-100 | 4% # 0.1% | 108.1 881.9 10 |
Quenching Buffer (QB) | 1M Tris-HCl pH8 1x PBS pH7.4 40 U/μl RNase Inhibitor | 200 mM # 0.16 U/ μl | 200 798 2 |
Resuspension Buffer (RB) | H20, molecular biology grade 1x PBS pH7.4 40 U/μl RNase Inhibitor | # # 0.16 U/ μl | 500 498 2 |
Specific Reagents
Vendor | Item | Cat. No. |
Sigma-Aldrich | DMSO (99.5 %) | D4540 |
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
Fixation Temperature and Time
1 h at 20ᵒC – for samples which are to be processed immediately after fixation
12-24 h at 4ᵒC – for samples which are to be stored
Sample Fixation (tested with 1 M DU145 cells/ml)
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.
11. Resuspend cells in 1 ml Resuspension Buffer (RB).
12. Pass cells through 40 μm strainer.
13. 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).
Fixed Sample Storage Guidelines
For short-term storage at 4ᵒC:
Add 0.05 volume of DMSO to fixed sample in QB buffer.
Store sample(s) at 4ᵒC max for 1 week.
For long-term storage at -80ᵒC or shipment on dry ice, proceed as follow:
Add 0.05 volume of DMSO to fixed sample in QB buffer (step 9, e.g. 50 µl DMSO to 1000 µl fixed sample). Gently pipette mix 5x.
Add 50% Glycerol for a final concentration of 10%.
Store at -80ᵒC for up to 6 months.