Cell Sorting & Analysis
General Requirements for Analyzers and Sorters
In this section you will find information for preparing your samples for flow cytometric analysis and also for getting the best possible outcome from your cell sorting experiement.
We strongly reccommend that you filter your cells prior to running on any of our instruments, there are various filter options available, please discuss with a memebr of the core facility. This is a great article on ways to prevent clumping:
Running correct controls are very important, you should come with at least the following:
- Biological Controls
- FMO Controls for multi-color experiements to set Negative gates
- Compensation Controls
- QC Controls such as beads to take into account instrument changes over time.
- Proper Panel Design will also ensure you produce excellent data!
The flow core strongly reccommends that you use a Live/Dead marker in all of your panels.
The core reccommends the following software to aid you in your flow analysis, we also provide two offline workstations free of charge with most of this software installed.
FCS Express 6
FlowJo (including tSne and vSne plugins)
FACS DiVa 8.1
Emory FlowJo Licence Page
For more information on FlowJo licencing please contact Kiran Gill (firstname.lastname@example.org) or John Altman (email@example.com). Yerkes Flow Core manages this, please direct questions to them.
You can use the following tubes on each of our instruments:
- FACSymphony / LSR II - 5mL 12x75mm Polystyrene Round Bottom Tubes.
- ImagestreamX MKII - 1.5mL Eppendorf Tubes for Unfixed Cells.
- ImagestreamX MKII - 1.5mL Siliconized Eppendorf tubes for Fixed Cells -> Beta South T-3406-250.
- Cytoflex - Plates Only - 96-Well Flat, Round or V-Bottom Plate.
Guidlines for Getting a Good Sort
Cells – How many do I need?
To answer it we need to know the following information:
- What is the approximate percentage of the population(s) you wish to sort?
- How many total cells do you want back?
- Do you want stringent purification or enrichment with great recovery?
- Do you have large or fragile cells?
All of these parameters influence the yield and purity of cell sorting.
For example: Let's say the subset you want sorted is 20% of the total and you need 2 million cells for your experiment. In theory you would need to run 10 million cells through the sorter (10 million x 20% = 2 million). However, the actual yield is usually 75-95% of this theoretical yield, due to abort rates (caused by sort conflicts) and also the quality of your sample. Therefore, we recommend that you bring 25-50% more cells to the sorter than you would need if the actual yield were 100% (based on the abundance of your target cells).
Time – How long will it take?
The answer to this question depends primarily on your goal (enrichment vs. stringent purification), the nature of your cells (fragile cells or large cells need to be sorted at lower pressures and speeds with a larger nozzle), and the concentration of your sample (more dilute samples will take longer to sort).
Filtering – Do I need to filter my cells?
It is REQUIRED that your cells be filtered through at 40um – 70um nylon mesh, preferably right before running on the sorter.
Re-suspension – What do I re-suspend my cells in for sorting?
After the final wash, cells should be re-suspended at concentrations of 20-30 million (for primary cells) or 5-10 million (for cell lines) per mL in BD 5mL 352063 Polypropylene tubes or 15mL BD 352196 Polypropylene tubes.
We recommend the following sort buffer for re-suspending your cells in:
2% FBS or BSA
25nM HEPES at a pH 7.0 (Stablizes Cell Mebranes)
EDTA if your cells are sticky (1mM is usually fine)
10units/mL of DNASE if there is high cell death.
We highly recommend using a Live/Dead Stain when cell sorting.
Collection Media – What media should you sort into?
We recommend that you sort into culture media with at least 20% FBS. For RNA/DNA PBS and if you have cells that are very fragile FBS only. Please discuss with the Technical Director for the best medium to sort into.
All users are required to download, fill out and bring the Pediatric Research Alliance Flow Cytometry Facility Biosafety Form to the Cell Sorting Facility prior to their sort being performed.
As of the May 1, 2012 all users that are requesting cell sorting are required to download the Cell Sorting Guidelines from the EHSO. A Risk Assesment must be provided to the Flow Cytometry Facility before any sorting can be performed, a template is provided within the EHSO guidelines.
We can sort from the following sample tubes:
- 5mL 12x75mm Polystyrene or Polypropelene Round Bottom Tubes.
- 1.5mL Eppendorf (with the lid cut off).
- 15mL Falcon Tube.
- 1mL micro-tubes.
We can collect into the following tubes/plates:
- Up to 4 5mL 12x75mm Polypropelene Round Bottom Tubes (not Polystyrene due to static charge accumulation).
- Up to 2 15mL Falcon Tubes.
- Up to 4 1.5mL eppedorf tubes with their lids cut off.
- Various Plates - 96-well, 24-well, 12-well, 6-well plates, Teraskai Plates.
- Petri Dish.
- Micrscope Slides.
If your collection device is not listed here please contact the core facility staff, we may just be able to sort into it!!
- 5 Essential Calculations For Accurate Flow Cytometry Results
- Antibody Titration
- Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages
- Basic Protocol for Staining and Compensation
- Creating a Template in DiVa
- Flow Cytometry Controls Paper