SHC Flow Cytometry Core
Affiliated with University of Cincinnati
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Guidelines for preparation for cell sorting on the SHC FACS Vantage.

 

 

Provide the proper information prior to scheduling your appointment:

 

1. What type of cells do you wish to sort (lymphocytes, cultured cells, adherent cells, primary cells etc?)

 

2.  What is the species of origin of the samples (human, murine, etc.)

 

3.  What are the biohazards associated with the sample? (Are these cells infected with a known virus?)

 

4.  What is the approximate initial concentration of the population you wish to sort?  (i.e. are the cells of interest 50% or 10% etc. of the total population).   If the population is extremely rare, you may wish to consider performing a bulk sort first (e.g. magnetic beads) in order to concentrate the population of interest prior to flow sorting.

 

5.  What is the size of the cell to be sorted (10µm, 20µm, etc.)?

 

6.  What fluorophores are you using to label your cells or antibody?  It is important to bring the proper controls for instrument set up! Please ask if you don’t know what control to bring.

 

7.  How many cells do you wish to collect?  The percentage of the population of interest, the cell concentration, and the desired number of cells recovered determine the length of your sort.

 

8.  What do you want to collect your cells into?  The FACS Vantage is capable of collecting samples in 5ml tubes, 15ml tubes, or 96 well plates.  Different collection strategies require a slightly different instrument set up.  With slight modification, the Vantage can accommodate 1.5ml Eppendorf tubes.

 

9.  Will your samples require cooling?  The FACS Vantage is equipped with a water bath that can heat or cool the cell sample and sort collection tubes.

 

 

 

Prepare the samples for sorting:

 

Sorting requires single cell suspensions that have a high viability and are not too concentrated.  A clog in the sort nozzle is catastrophic, so every effort should be made to keep the cells from clumping.  The following should assist you in preparing good samples for sorting.

 

1. Count your cells and assess the viability.

 

2. Keep the cells (typically hematopoietic or those grown in suspension) at a concentration no greater than 1 x 107 cells/ml.  Sticky cells (or adherent cells) should be 3 - 5 x 106 cells/ml.  The cell concentration of cells for a plate sort should be approximately 1.5x106 cells/ml.  Samples to be sorted should be placed in BD 12x75mm falcon tubes (BD Falcon 352054).

 

3. Filter the cells (40um filter, usually nylon mesh) prior to bringing them to the flow lab.  An additional filtration may be required immediately prior to sorting if you are bringing multiple samples to the lab (the cells may settle and clump while waiting to be sorted). 

 

4. Keep the protein concentration in your cell suspensions to a minimum: 0.1%-1% BSA or FBS.

 

5. Use a buffer that is Ca++ and Mg++ free (see below for an example of a buffer formulation).

 

6. Add 1-5mM EDTA to the cell suspension to keep cells from sticking together.

 

 

Prepare for sample collection:

 

The FACS Vantage is capable of collecting samples in 5ml tubes, 15ml tubes, or 96 well plates.

                                                                                                             

1.  It is strongly recommended that your collection buffer contain a high concentration of serum (at least 10%, you may use up to 100% serum) and antibioticsIt is nearly impossible to keep cells sorted in a jet-in-air sorter sterile without antibiotics.  

 

2.  Prepare your collection tubes by adding high protein collection media to each tube.  Place approximately 1ml of media in the 5ml tube and between 3 and 5mls of media in the 15ml tubes. Place 100ul of media in each well of a 96 well plate.

 

3.  Please bring extra sort buffer, collection media, and collection tubes!  Occasionally, samples require dilution prior to sorting.  Also, it may be necessary to use more than one collection tube for each sample to be sorted.

 

 

Common sample buffer for cells to be sorted:

 

1X PBS (Ca++ and Mg++ free)

25mM HEPES  (pH 7.0)

1% (or less) heat inactivated FBS or BSA (depending on cell type), if your cells can tolerate it 0.1% tissue culture grade BSA is best

EDTA (up to 5mM)

Or Hanks Balanced Salt solution (Ca++, Mg++ free, and phenol red free) containing same supplements as above.

 

Commonly used collection medium:

 

1.     1.  Culture media containing at least 10% serum + antibiotics-50% serum probably optimal for most cell types

2.    2.  PBS

3.    3.  RNA Lysis buffer