CELL PREPARATION FOR FLOW CYTOMETRY CELL SORTING Sort parameters
Described in the sections below are some general guidelines for preparing samples for flow cytometry particle sorting. For live cell sorting, I suggest a cell density of 2X10^6 cells/ml. The percent of cells expressing fluorophore of interest (i.e. GFP) will be a determining factor as to how many mL of cells suspension is needed for the sorting process. Another important factor is how many cells you want to collect from the sort.
- Fluorophore(s) of interest – excitation/emission
- DAPI –387nm/447 nm
- GFP – 488 nm/509 nm
- YFP – 500 nm/542 nm
- PE – 496 nm/578 nm
- TRITC – 543 nm/593 nm
- Texas Red – 562 nm/624 nm
- dtTomato – 554 nm/581 nm
- mCherry – 562 nm/641 nm
- Cy3 – 531 nm/593 nm
- Cy5 – 628 nm/692 nm
- What percentage of cells are expressing fluorophore(s) of interest?
- How many cells needed from sort?
- Harvest cells and resuspend in flow cytometry friendly media with 2X antibiotics at approximately 1-2x106cells/mL.
- Flow friendly media – needs to a clear buffer, listed below is the recipe for basic sorting buffer
- Supplement the buffer with 2X antibiotics (i.e. penstrep) – this will help with prevention of contamination during the sorting process
- samples need to be in Faclon 5-mL sterile tubes – bags of tubes are in the flow cytometry facility, see April for access. Also, I suggest keeping the cells on ice when transporting and sorting.
We need to perform a pre-sort analysis to determine the percent of cells expressing the fluorophore of interest. Expressing cells will be gated for the population of interest to be sorted. Unstained or non-expressing cells need to be used as a negative control. For the pre-analysis you will need 2 tubes:
Live Cell Sorting
- unstained / non-expressing cells ~ 300 uL
- cells with expression of interest (i.e. EYFG) ~ 300 uL
Now that the population of interest has been gated, now we can sort the cell suspension. The most challenging factor is contamination. In order to prevent contamination, the sort will be performed twice (with 2 separate samples). Each sort will be sorted into 2 tubes (depending on expression levels and cell density), therefore after the sorting process there will be 4 tubes with cells for plating. It is best to sort the cells into tubes with growth media with 2X antibiotics. The sorting process is harsh on cells and recovery seems to be quickest when cells are sorted into tubes containing the normal growth media. With all solutions involved in the sorting process it is best to use 2X antibiotics to prevent contamination.
For the sorting you will need 6 tubes:
- cells in flow friendly media sample #1 ~1500 µL @ 2x10^6 cells/mL in cytometry tube
- cells in flow friendly media sample #2 ~1500 µL @ 2x10^6 cells/mL in cytometry tube
- Cytometry tube with growth media with 2x antibiotics #1
- Cytometry tube with growth media with 2x antibiotics #2
- Cytometry tube with growth media with 2x antibiotics #3
- Cytometry tube with growth media with 2x antibiotics #4
All of these parameters can be modified for optimization based on users' research needs.Post-Sort Analysis
Representative samples from post-sorted cells will be ran to determine expression percentages of cell population. Basic Sorting Buffer Recipe
For Clean Lymphoid Cells
- 1x Phosphate Buffered Saline (Ca/Mg++ free)
- 1mM EDTA
- 25mM HEPES pH 7.0
- 1% Fetal Bovine Serum (Heat-Inactivated)
- 0.2um filter sterilize, store a 4oC
: The buffer can be simplified to HBSS with 1% FBS. The additional cations in the recipe promote better viability. Since these cells are not prone to clump, the lack of EDTA is not a problem.For Sticky Cells
: Raise the concentration of the EDTA to 5mM and use FBS that has been dialyzed against Ca/Mg++ free PBS. Some activated cells become clumpy and the chelators (EDTA) help reduce cation dependent cell to cell adhesion.For Adherent Cells
: In order achieve good single cell preparations, one must start at the moment of detaching your cells from the plate. Typically, the trypsin (or other detachment buffer such as Accutase) is quenched with culture media or a PBS/FBS buffer. This is problematic because it reintroduces the cations that facilitate the cells reattaching to the plate (or each other). One must use a cation-free FBS buffer in order to stop the detachment. Additionally, the level of EDTA can be increased if necessary (but too much EDTA can be deleterious).For Samples with High Percentage of Dead Cells
: If there are a large number of dead cells in the prep, it is likely that there is soluble DNA from the dead cells that will come out of solution. This DNA will start to coat the cells and and lead to severe clumping. The addition of 10U/mL DNAase II to the buffer recipe will help reduce DNA associated clumpiness.