Sort Guidelines and FAQ
at the BCM Cytometry and Cell Sorting Core (CCSC) Facility
Sample Preparation
 Protein conc. of 0.1%-1% in media ie.HSA, hAB serum, or BSA (absolute max.
conc. 2%)
 HEPES buffer recommended
 Calcium free media can help reduce clogging and increase efficiency
 Cell Concentration 10 to 20 x 106 cells/mL
 Filter cells using 35-40µm filter (35µm filter-cap tubes from BDFalcon
Product#352235 available for purchase from the CCSC for $25/25 tubes)
 Set-up samples and controls into one of the following tubes with lids: 15-mL
conicals, 5-mL round-bottom tubes, or 1.0- 1.5 mL micro-tubes
 Maximum sample volume of 3.5mL per 5-mL tube and 13mL in a 15-mL conical
 Catch tube/plate/slide Information
o Assure you have appropriate catch media for your sample including a noncarbonic buffer
 Protein Concentration
 Type
 Volume
 Consider whether Ca++/Mg+ required for cells
 PBS or RPMI
 Recommended catch tube media volume
 0.5-1mL in 5-mL Falcon tube
 1-4mL in 15-mL Falcon tube
 100 uL in 1.5-mL Eppendorfs or 96-well plates
 1mL in 12-well plate
 0.5 mL in 24-well plate
 250 uL in 48-well plate
o Size/Type of catch tube
 15-mL conical tube (max 2 tubes)
 5-mL round bottom tubes (max 4 tubes)
 1.5-mL Eppendorfs
 Plates (6 wells to 96 wells)
 Slides- frosted end or standard
 Protein coated plastic can reduce chances of small cell populations
drying out on side of tube during sorting
o Correct Tube numbers including extras in case multiple tubes for
individual populations are needed.
 Adequate Labeling of sample tubes, catch tubes and media
What you need to know before sorting:
 Size of your cells
o Why?
 Increase viability
 Nozzle size uses cell size Optimal Resolution
 Less splattering of stream
 Decrease chances of clogs
 Increases purity
o How?
 Microscope
 ViCELL located at CCSC- (sizing should be done at least one day
before your first sorting appointment at no charge)
o Cell size vs. Nozzle Size
 5-15um = 70um nozzle
 16-25um = 100um nozzle
 25-32um = 130 nozzle
 Cell Adhesiveness
o Why?
 Prevent clogs- frequent filtering and vortexing throughout sort can
minimize clog formation which would slow or stop sorting
 Help determine sorting media
o How?
 Microscope or Vi-CELL
 General nature of cells, i.e. stick to plastic
 Trypsinization required for longer periods
 Total cell number
o Why?
 To achieve optimal sorting concentration- minimize clumping and
prevent clogs
o How?
 Microscope
 ViCell at CCSC



Relative experimental expectations
Biohazards (Must inform sort operator in advance of potential biohazardous
material)
Proper controls for your experiment- See next page
Frequently Asked Questions (FAQ)
Q.
How many cells can I get?
A.
It is entirely up to your experiment. This depends on percent expression and
relative efficiency allowed by sample.
Q.
How fast can the machine go?
A.
As fast as you want but to ensure cell viability, purity and maximum yield we
adjust speed to sample efficiency and overall durability. A good range would be
from 3 million to 10 million total cells per hour on the 130µm nozzle and 50
million to 70 million total cells per hour on the 70µm nozzle with high pressure.
Q.
Can I just drop off my cells and then go?
A.
This is highly frowned upon. Since this is your experiment you presence is
extremely helpful during set-up. Once set up is complete you can go as long as
you leave a contact number.
Q.
What controls should I set-up?
A.
Controls are needed every sorting appointment.
For a GFP sort or other FP, a negative cell line sample is greatly appreciated,
ideally one transfected with a non-fluorescent protein- especially for initial set-up
For a simple one-color experiment, an unstained control is fine. Multi-color
experiments require an unstained control and single-stained controls for each
fluorophore. For optimal instrument set-up and experiment precision, we strongly
recommend the use of isotype controls which use antibodies with different source
and target than your cells to show non-specific staining (background). See below
an example chart of isotype controls. For more information on isotype controls
contact the CCSC at sederstr@bcm.edu.
Q.
Should I put PI in all my control tubes?
A.
Ideally only the PI single positive control tube would have PI in it. Always have
at least one control tube without PI.
Example Isotype Controls Chart:
Tube 1
Tube 2
Tube 3
PE
Non-specific
Positive
Non-specific
FITC
Non-specific
Non-specific
Positive
Tube 4
Positive
Positive