We recommend that cells are fixed prior to isolation in order to preserve their transcriptomic state (importantly. Importantly, dead cells will not be compatible with LUTHOR HD library preparation).
Cell fixation guidelines are below which have been validated internally , which have been validated internally, and are outlined below.:
Buffers
Buffer | Stock | Final concentration | To be added |
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 1x PBS pH7.4 (10010-023) 40 U/μl RNase Inhibitor (Y9240L) | # # 0.16 U/ μl | 500 498 2 |
Specific Reagents
Vendor | Item | Lot |
Sigma-Aldrich | DMSO (99.5 %) | D4540 |
ThermoFisher | Glycerol* (99%, MBG) | J61059 |
*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). 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.
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 and determine viability of cells. Strongly recommended to stain cells with a fluorescent dye An efficient fixation protocol should give >95% non viable cells. We recommend staining with fluorescent dyes such as Acridine Orange /and Propidium Iodide . >95% of fixed cells appear non-viable(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.
Fixed Sample Storage Guidelines
Fixed samples can be stored at 4ᵒC for up to 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
ulµl fixed sample). Gently pipette mix 5x.Add 50% Glycerol for a final concentration
.concentration of 10%.Store at -80ᵒC for up to 6 months.