Samples suppose to be single cell suspension (SCS) only (i.e. no clumps). Use all applicable Sample Preparation Tips
If you have extensive cell death in the sample, add DNAse (see Buffer Suggestions). It will not only decrease clump formation in the sample, but also will decrease viscosity of the single cell suspension buffer, by digestion of the free DNA. Sort purity and yield could go substantially up after DNAase treatment
Add the 10-25mM of HEPES, pH range 6.8 – 8.2 (for example: HEPES buffer solution 1M in H2O, Sigma-Aldrich Co., Cat# 83264-100ML-F) to your buffer. Addition of HEPES will significantly increase the buffer capacity of the original sample buffer. Buffer capacity of the common phosphate and carbonate buffers gets compromised by high pressure within the instrument during the cell sorting procedure
Provide compensation controls:
If cells are used as single color controls
Unstained cells
Titration of the Dead Cell Exclusion dye on the “partially killed” cells
All separate single color stained cell controls
Preferable concentration of the controls is around 1 x 106cells/ml, volume 0.5-1.0 ml
If beads are used as single color controls
Unstained Cells
Preferable concentration of unstained cell control is around 1 x 106cells/ml, volume 0.5-1.0 ml
Provide gating controls – Fluorescent Minus One (FMO) controls, as proper way to evaluate the background in certain channel and set sorting gates appropriately
Sample concentration for the sort is 1-20 x 106 cells/ml (dependent on the cell type and the nozzle size)
Provide extra 15 ml of the buffer you used for the cell samples
Provide 1 ug/ml stock solution (1 ml) of the non-fixable Dead Cell Exclusion dye on the first experiment. On the consequent experiment, bring 15 ml of buffer containing 5X of the non-fixable Dead Cell Exclusion dye
Bulk Sort – Provide collection tubes for the sorted cells. Tube size depends on the expected number of post-sorted cells. Note: Concentration of post-sorted cells is between 0.3 – 1.0 x 106 cells/ml and is depends on the nozzle size
Tube types:
Screw Cap Micro Tubes (up to 4-way sort)
1.5 ml – sterile (Cat#89499-612, VWR)
1.5 ml – low retention tubes (Cat#20170-650 / 76332-068, VWR)
2.0 ml – sterile (Cat#89499-620, VWR)
12 x 75 mm Tubes (up to 4-way sort)
Polypropylene – best choice
sterile with snap cap (Cat#1495911A, Fisher Scientific; or Cat#352063, BD Biosciences)
Polystyrene – poorer choice, but still possible
non-sterile (Cat#352008, BD Biosciences)
sterile with the cap (Cat#352054, BD Biosciences)
15 ml Tubes – any type (up to 2-way sort)
50 ml Tubes – any type (up to 2-way sort)
Tube pre-treatment:
In order to prevent cells sticking to the sides of the tubes, pre-coat the tubes, filling them with 10x BSA solution (1%)
Keep filled tubes inverted for at least 30′ prior sort
Low retention tubes (Cat#20170-650 / 76332-068, VWR) can be used without additional pre-coating
Solution to sort into:
TRIzol LS reagent (for RNA extraction. Volume of TRIzol LS:Cells=3:1. For example, 750 ul of TRIzol LS + 250 ul of sorted cells)
PBS-based buffer
PBS/HEPES/BSA (see buffer suggestions)
Any specific solution (for example, PCR mix)
none
Tube special characteristics depend on the sorting purpose:
For sterile sort tubes should be also sterile
For RNA work tubes should be RNAse free
For Western blot tubes should not be treated with external protein
Processing of the post-sort samples:
All bulk-sorted samples for the cell culture should be washed before plating
We recommend washing each sample at least twice with the 3-5X antibiotics in the wash medium
After appropriate washing steps (see above) cells should be re-suspended in the complete essential medium with the 1X antibiotics
Sort using ACDU (automated cell deposition unit):
Receptacles:
Multiwell plates:
96 well plate
384 well plate
PCR Tubes in stripes
Microscope slides
Etc.
Solution to sort into – depends on the purpose of the sort:
Complete medium for cell growth
TRIzol LS reagent (for RNA extraction)
PCR mix
Etc.
Sort setup specifics:
Day before the sort FCRC Staff should be provided with several empty plates (or other receptacles) of the type planned to be used for the sort
Plates (or other receptacles) for the cell sort should be brought to FCRC at the time specified by FCRC Staff (when sort is completely pre-set using empty receptacles)