Flow Cytometry Facility

Helpful Information

Basics of High-Speed Cell Sorting on the Cytomation MoFlo
Post-Acquisition Analysis Software – FlowJo, Summit, WinMDI
Fluorescence Spectra of Commonly Used Dyes
Premiere Website for Flow Cytometry
Compensation Tutorial Fluorescence Conjugation of Antibodies
How to attribute the flow cytometry equipment in your publication

BASICS OF HIGH-SPEED CELL SORTING on the Cytomation MoFlo

HOW LONG WILL IT TAKE ME TO SORT?
Calculation for the 70 micron nozzle (lymphs, pbmc, bone marrow, yeast):
(For most cell lines, a 100 micron nozzle is needed, which reduces throughput by at least 70%)
Nominal maximum throughput (70µ) = 20-25,000 events/second ~72 - 90(10)⁶events/hour
Net throughput (corrected for normal recovery/yield estimates) = 70% ~50-63(10)⁶ events/hour
Estimated throughput = 50(10)⁶cells/hour (50 million cells per hour that can be run through the machine)
For most cell lines, a 100 micron nozzle is needed, which reduces throughput by at least 70% - adjust calculation accordingly.

Collection rate, 70µ (maximum) = (viability) x (expression) x 50(10)⁶ events/hour
EXAMPLE:
Attempting to sort out a sub-population (5%) from your cell line
The number of cells you can provide is unlimited, and the viability is 90%.
A pure, sorted population of at least 5(10)⁶cells needs to be recovered.
Given the estimated sorting capabilities (see the calculation above), estimate sorting time as follows:
Collection rate (maximum) = 0.9(viability 90%) x 0.05 (expression 5%) x 50(10)⁶ events/hour
=(0.9) x (0.05) x 50 million cells/hour =(.045) x 50(10)⁶ = 2.25(10)⁶ = 2,250,000 per hour.
100 micron nozzle reduces by at least 70% - adjust calculation accordingly.

Recovery of  5(10)⁶ total =[5(10)⁶ ÷ 2.25(10)⁶] = 2.22 hours of sorting, at minimum Add an additional 45 minutes to sorting time to set up the machine.
However, please assume that something might go wrong, and you will need twice the number of cells, and it will take twice as long.
REMEMBER:  4 SEPARATE POPULATIONS CAN BE COLLECTED AT THE SAME TIME.

FOR ANY SORT, YOU WILL NEED TO BRING THE FOLLOWING:
1. Control(s):
a. Negative control
b. Compensation controls if needed
(If you have any questions about appropriate controls, please ask your friendly flow cytometry technician)

2. Sort sample(s)
Bring controls and samples in 12 x 75 mm sterile; snap-cap; POLYPROPYLENE tubes, with no more than 5% serum.
The control(s) only needs to be 300 uL final volume, with at least 300,000 cells.
Sample should be re-suspended at a concentration on 30-40(10)⁶ cells/ml.

3. Collection tubes (4 separate populations can be collected at the same time)
For each tube for collecting sorted cells, fill the tube ¼ full with 50% serum/media. You may use either 12x75mm tubes or 15 ml tubes.
(12x75mm tubes are needed for 4-way sorting - fill the tube ¼ full with 50% serum/media.)
Cells to be collected exit the machine at a concentration of ~1,000,000 cells/ml using the 70 micron nozzle; 500,000 cell/ml using the 100 micron nozzle. You can now calculate how many collection tubes you will need for each population collected.
(Example: For 70µ nozzle, you place 4ml media/serum in the bottom of a 15ml tube, leaving 11 ml empty volume for collecting cells = 11ml x 1,000,000 cells/ml = 11(10)6that may be collected in that tube.)

FOLLOWING THE SORT
For maximum recovery, fill collection tubes to the top, let sit 30 minutes before spinning them down.

BASICS OF CELL SORTING on the EPICS Elite
HOW LONG WILL IT TAKE ME TO SORT?
Nominal maximum throughput = 4-5,000 events/second à14.4-18(10)⁶events/hour
Net throughput (corrected for normal recovery/yield estimates) = 70% à10-12.6(10)⁶ events/hour
Estimated throughput = 10(10)⁶cells/hour (10 million cells per hour can be run through the machine)

Collection rate (maximum) =(viability) x (expression) x 10(10)⁶ events/hour
EXAMPLE:
Attempting to sort out a sub-population (5%) from your cell line
The number of cells you can provide is unlimited, and the viability is 90%.
A pure, sorted population of at least 2(10)⁶ cells needs to be recovered.
Given the estimated sorting capabilities (see the calculation above), estimate sorting time as follows:
Collection rate (maximum) = 0.9(viability 90%) x 0.05 (expression 5%) x 10(10)⁶ events/hour
 =(0.9) x (0.05) x 10 million cells/hour =(.045) x 10(10)⁶ = 0.45(10)⁶ = 450,000 per hour.

Recovery of 2(10)⁶  total = [2(10)⁶ ÷ 0.45(10)⁶] = 4.44 hours of sorting, at minimum.
Add an additional 45 minutes to sorting time to set up the machine.
However, please assume that something might go wrong, and you will need twice the number of cells, and it will take twice as long.
REMEMBER: 2 SEPARATE POPULATIONS CAN BE COLLECTED AT THE SAME TIME.
FOLLOWING THE SORT
For maximum recovery, fill collection tubes to the top, let sit 30 minutes before spinning them down.

Post-Acquisition Analysis Software – FlowJo, Summit, WinMDI (back to top)

FlowJo (Mac and PC) is the most commonly used post-acquisition software package, and may be used to analyze data files from any of the available instruments.  Wistar investigators may purchase this software through the Institute site license.   Please contact Jeffrey Faust for more information.

Summit (PC) is the free software running on the DakoCytomation instruments, and may be used to analyze data files from any of the available instruments.  Please contact Jeffrey Faust if you are interested in obtaining a copy.

WinMDI is a PC-Windows based, free post-acquisition analysis software package which may be used to analyze flow cytometry data files from any of the available instruments. Curve smoothing, re-gating of list mode data, and overlays are some of the available functions. Generated images may be cut-and-pasted into PowerPoint presentation software. To download the software, click on the following link:
http://facs.scripps.edu/software.html

Fluorescence Spectra of Commonly Used Dyes (back to top)

These links will give very useful visual packages of dye excitation and emission spectra, in order to determine whether or not certain dyes can be used alone or in combination on the available flow cytometry equipment:
http://www.bdbiosciences.com/spectra
http://probes.invitrogen.com/servlets/spectraviewer?fileid1=2761old

Premiere Website for Flow Cytometry (back to top)
The following premiere website addresses all aspects of flow (and some image) cytometry. The website provides useful links, as well as discussion groups where you can post specific questions and comments (check the “Cytometry E-mail” link). http://www.cyto.purdue.edu

Compensation Tutorial
Fluorescence Conjugation of Antibodies

This website has protocols concerning these topics related to multi-color flow cytometry, from Dr. Mario Roederer, a trendsetter in multi-color flow.
http://www.drmr.com

How to attribute the flow cytometry equipment in your publications (back to top)

Publications using data generated using equipment from the facility, which require a description in a “materials/methods” or similar section should use some variation of the following:

• Samples were analyzed using a BD LSRII (BD Biosciences, San Jose, CA)
  Or
• Samples were analyzed using a BD FACSCalibur (BD Biosciences, San Jose, CA)
  Or
• Samples were analyzed using a BD FACScan (BD Biosciences, San Jose, CA)
  Or
• Samples were analyzed using an EPICS XL (Beckman-Coulter, Inc., Miami, FL)
  Or
• Samples were analyzed using a DakoCytomation CyAn ADP (DakoCytomation, Inc., Fort Collins, CO)
  Or
• Samples were sorted (or analyzed) using a DakoCytomation MoFlo (DakoCytomation, Inc., Fort Collins, CO)

If you are uncertain of which instrument you used, or need more technical information, please contact the facility.  For publication purposes, the names of the operators are Jeffrey S. Faust, Daniel J. Hussey, or David E. Ambrose.