Catalog of Free Flow Cytometry Software

The original Cytometry software catalog was developed and managed by Dr. Eric Martz for several years. In cooperation with Dr. Martz, the site will now be managed by the Purdue University Cytometry Laboratories. Our goal is to provide comprehensive educational materials in all areas of cytometry and broadly in science where we can. If you want to contact Dr. Martz please do so.

This catalog strives to be comprehensive -- to include as much free flow cytometry software as possible. We have also added some demonstrations of commercial packages. Report comments, catalog omissions and corrections to


Problems getting program files? Here's HELP.


Purdue Free software is all listed on another page - you can go here to find Cytospec, or PlateAnalyzer


Walter & Eliza Hall Free Software is Here

Multi-Purpose Analysis Packages

Cell Cycle/DNA Analysis

  • IDLYK (Unix, VMS, PC/Windows, MacIntosh)

Cluster Analysis

Other Special Purpose

Data Transfer from Hewlett-Packard to PC/Mac

Miscellaneous PC Utilities

Miscellaneous Macintosh Utilities

WinMDI (PC/Windows)

Windows Multiple Document Interface

Best available for general purposes; colored regions, quadrant analysis, complex gating. Gorgeous graphics, 3D contour and dot plots. Created by Joseph Trotter of the Scripps Research Institute.

WinMDI (Windows Multiple Document Interafce for Flow Cytometry) Requires MicroSoft Windows 3.1 or better. Version 2.5 requires a 486 or better processor. If you need to run under Soft Windows on a Macintosh , get the special version that uses the 286 instruction set.

A free-standing slideshow is available which demonstrates WinMDI's graphics. This slideshow operates under DOS (it does not require Windows). It is available as a self-unzipping file, WMDISHOW.EXE. Running the show is very simple. Instructions are included.

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Median Fluorescence Intensity

Fast batch analysis of listmode files including graphics and overlays, fully corrected intensities, automatic peak detection, kinetics, calculated parameters, gate ratios. Spreadsheet-ready medians. Labels listmode files, converts to listmode or histogram data to ASCII for spreadsheets. Tutorial with sample data. (Does not do quadrant analysis.)

Works perfectly in Windows.
Full documentation and the program are available from the MFI Home page

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This is the only free cluster analysis software available for PC's. Cytometry 14:649-659, 1993. Author: Tom Bakker Schut, Netherlands. Richard Allen Cox is enhancing AUTOKLUS. For further information about the status of this project, see the Flow Cytometry Support Home Page.

Original free Schut AUTOKLUS program .
Information about the included program
Included source code
Included data files

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IDLYK (unix, VMS, PC, Mac)

General purpose program with many graph formats to facilitate the comprehension of multi-parameter data. Includes: 1D histos, lineplot, image display, dotplot, contours, surface plot, 3D dot plot, 3D iso-surface; kinetic display of mean, +/- 1, cv vs time. Also provides cell cycle analysis, neural net clustering, batch mode support, composite graph generation (from multiple files), up to 10 rectangular or irregular regions of interest for color highlighting between parameters or editing, and several forms of hardcopy output (PS, HPGL, PCL, GIF, PICT).

IDLYK is a free script for a commercial interpreter "IDL", which costs $1,500 per cpu (PC or MAC; more for unix/VMS). Many universities have existing IDL licenses on network servers (inquire from Jim Hill at Research Systems, Inc., the source of IDL,

Inquire from the author: Robb Habbersett, Los Alamos National Lab, NM USA;

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LAP (Unix, VMS)

Laboratory Analysis Program

Input limited to 1D and 2D histogram FCS data only (does not handle listmode data). Powerful and flexible general purpose data analysis and manipulation interpreter which uses FCS-type storage format. Does histogram overlays, 2D and 3D contour plots. Scripts exist for flow data which can be reprogrammed as needed. Request program from author: John Powell, NIH, Bethesda MD USA,

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General purpose with polygonal gates (mouse controlled), contour and density plots, histogram overlays, kinetics vs. event number and file slicing. New parameters can be created by transformation and division. Supports only one printer, the HP Paintjet. Comp Biomed Res 27:83-96, 1994. Author: Tom Bakker Schut (see under AUTOKLUS) states that no further development is expected.

Editorial comments: WinMDI is preferable for most purposes, although ANALYSE has a few unique features. ANALYSE is ten to five times slower than WinMDI or MFI to read a listmode file. Because of its Windows interface, WinMDI supports a much wider range of printers. WinMDI has more flexible gating logic, better numeric results for histograms and marked segments of histograms, better control of color or line type for overlayed histograms. -Eric Martz

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FACSOVL (PC/Windows)

Displays Coulter XL histogram files while smoothing, scaling, changing resolution; overlays up to 8; outputs to ASCII for spreadsheets. Author: Frederik Lindberg, Wash U, St Louis MO USA.

Get self-unzipping program FXOVLZIP.EXE by ftp.

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PC ANalysis

This is an old but elegant scatter gating and quadrant analysis program provided to the public domain by Becton-Dickinson. It determines percentage positive cells from FCS1.0 files for one, two, or three color work. It supports polygonal gates, requires EGA, and puts out summary files in ASCII database format (e.g. suitable for Lotus). After you have set the gate (typically on FSC vs. SSC or scatter vs. FL3 = propidium iodide), and set a division line for positive/negative on each color (it will automatically set the division for 99% negative on your control cells!), it processes all files in the current directory without further intervention. However, it displays the dot plot and histograms for each file for a user-specified number of seconds during which pressing any key allows modifications. Simple-minded but actually quite slick. Most VGA screens support the EGA it requires.

PeCAN was written by Morgan Conrad and Ravi Mhatre while at B-D. C source code is included.

Get PeCAN from the CD-ROM.
Get PeCAN source code from the CD-ROM.

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MFI/FCS Verification Suite

This verification suite provides tools for generating FCS (Flow Cytometry Standard format) data files with known contents. It is provided as a companion to MFI but is suitable for verification of any program capable of reading generic FCS files. Tests with synthetic data represent one of several methods of verifying accuracy of results.

The included data files can be used under any operating system, but the programs run only under PC DOS (or in a DOS window under MS Windows). However, the programs can be used under DOS to generate additional data files which can then be moved to any operating system.

The suite includes the program A2FCS.EXE which converts ASCII spreadsheet-style data files to FCS. It can handle 8- or 16-bit data, and can mark the data as log-acquired. Programs are provided to generate columns of random numbers; the results (medians etc.) can be independently obtained by the flow cytometry program of your choice, and by generic spreadsheet software for verification. The programs can generate arbitrarily large FCS files of repeating cycles of random numbers (hence with known medians, etc.). MFI has been tested using this strategy for up to 2,000,000 events of 3 parameters each. (Tools are also provided for generating data to display the greeting of your choice as a dot plot.) Further details are available in the MFI/FCS Verification Suite documentation.

MFI/FCS Verification Suite
Version 1.1 released 8/16/96 corrects the $BYTEORDer which was reversed; thanks to Richard Carson.

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This program converts numeric data in an ASCII file to an FCS file which can be read by flow analysis software. The ASCII file can be created by a spreadsheet or any other means. Since MFI, WinMDI, and other software can convert FCS files to ASCII files, one could edit the data in any way desired and then re-convert to FCS. This utility permits test data generated mathematically to be used to test flow analysis software. Author: Joseph Trotter, Scripps Research Institute, La Jolla CA.

Note: an alternative implementation, A2FCS.EXE, is available in the MFI/FCS Verification Suite.

Get the Text2FCS program from the CD-ROM or by ftp from the Scripps Institute.

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FACSLOG is suite of 4 programs to manage user access of HP 9000/310 and 9000/340 computer systems. It may be started automatically or manually after the computer has booted. Users will be required to "log-in" with their initials and a password. An optional "welcome" or "news" screen is displayed at login. Log-in and log-out dates and times for each user are recorded in a data file, which may be searched and printed by date and the users' "group" assignment (such as lab affiliation or billing account number). This data may also be saved in a file suitable for import in spreadsheet or database programs. Only "System Administrators" may enter and remove users or make changes to the system-use log file. FACSLOG was created by Neal A. Benson, University of Florida, Gainesville FL USA,

Get FACSLOG from the CD-ROM.

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PC printing on an IEEE-488 GPIB Paintjet

Hewlett-Packard (HP) computers sold with FACS instruments were often bundled with Hewlett-Packard Paintjet color printers. These printers typically have only an IEEE-488 GPIB connection (rather than the standard Centronics parallel printer connection expected by a PC). Nevertheless, if you have an IEEE-488 GPIB card in your PC (see HPPCLINK), you can not only transer files between the HP and PC, but you can print directly from the PC to the Paintjet. Here you can get an ASCII text file, PAINT488.TXT, containing detailed instructions on how to do this.

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Purdue Utilities (PC DOS)

The Purdue Utilities are a family of programs which include the following:

GENP takes a two parameter correlated FCS histogram, and generates a list of 'points' and 'weights' from it. It is designed to generate a list of scatterplot points to be read by some other analysis, statistics, or graphics program.
GENP documentation.

KEYS is used to generate a listing of all the keyword-value pairs in an FCS file. This includes the FCS TEXT and ANALYSIS segments, but not any private segments. [MFI displays text block keyword-value pairs to the screen from a point-and-shoot menu of filenames; it does not display the analysis block.]
KEYS documentation.

LDATA reads an FCS histogram file and writes its raw data segment to the screen or to an ASCII file. It is used when it is convenient to have histogram data in a human readable format, or when the data needs to be read by a standard analysis or statistics program. [MFI can write a gated, smoothed ASCII histogram data file from the list mode file.]
LDATA documentation.

LLDATA is used to generate a text listing of the data in a listmode file. [MFI can produce a gated ASCII file of list mode data from an FCS list mode file.]
LLDATA documentation.

LOOKUP opens FCS files, and retrieves specific keyword values.
LOOKUP documentation.

SETKEY is used to change the value of a keyword in an FCS file or to add new keywords to the file. [MFI can change values of keywords which are not critical to data integrity, or add new keywords and values.]
SETKEY documentation.

Authors: J. Paul Robinson and Steve Kelley, Purdue Univ., West Lafayette IN USA.

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JCS Family of FCS1.0 Utilities (PC DOS)

(John Curtin School)

This family of utilities includes FCSVIEW, a simple program to plot histograms out specifically on a HP pen plotter (in HPGL). Another utility program reverses the byte order of the binary listmode data in FCS1.0 files.

Inquire from Geoffrey Osborne, John Curtin School of Medical Research, Australian National University, Canberra

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FCS Assistant (Mac)

A macintosh utility to convert FCS1.0 and 2.0 files to text. It also adds calculated ratio, and time parameters and saves in FCS2.0. The program outputs three types of text file: raw list mode data, histogram data, and line graph data (eg mean Calcium v Time). It can handle up to 16 parameters, and allows the user to delete redundant parameters to make space. B-D vax, HP, and Mac files are opened as are Coulter IBM files. At present only Mac format FCS2.0 files are exported, but in future versions the program will save in FCS1.0 formats (LYSYS, consort 30, FACScan etc) to allow users of older systems access to ratio and time.

(Editor's note: MFI, a DOS package, also calculates and normalizes the ratio of two existing parameters, and plots any parameter vs. time or pseudo-time [event number]. It can put the results out into ASCII listmode or histogram files. Its companion program A2FCS can convert the ASCII listmode with calculated parameter into an FCS2.0 file. See also Refresh Ratio.)

Compiler/Language: Written in Think Pascal 4.0.2 for the Macintosh using ThinkClass Library version1.1. Source Code: "Too Embarrassing to Distribute". Author: Ray Hicks Get FCS Assistant from the CD-ROM.

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DropPrint (Mac)

Prints cytometer settings as recorded in files acquired with CellQuest (tm Becton-Dickinson) version 1.1 or greater, from FACScan, FACSort, or FACSCalibur instruments. Provided without warranty by its author, Ted Kunich, Get DropPrint from the CD-ROM. NOTE: You will need a version of Stuff-it Expander to unpack this program.

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MacNormalize (Mac)

Normalizes flow cytometric immunophenotyping data to a biological standard. This program is under development. Contact Gary Salzman, Los Alamos National Laboratory, Los Alamos NM USA.

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Refresh Ratio (PC/Windows)

This PC/Windows program refreshes the existing ratio of two user-specified parameters in LYSYS II listmode files (B-D FACScan, FACStar), or creates a new ratio parameter. It is untested on other FCS2.0 files. Compatible with linear or log data, 256 or 1024 channel resolution. In some instruments, the hardware/software is not fast enough to generate a correct ratio value for all events; Refresh Ratio fixes this problem.

The author is Ron Hoebe at the University of Amsterdam, Netherlands, Report problems or incompatibilities to him. (The program was written in Visual Basic 3.0 for Windows. Source code is not provided.)

Catalog editor's note: MFI (see elsewhere in this catalog) can calculate the ratio of two user-specified parameters, and rescale the result in several optional ways. Unlike Refresh Ratio, MFI calculates the ratio during analysis, and does not write it to the listmode file.

Get Refresh Ratio, or visit their home page.

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Copy from an HP diskette on a DOS computer

Self extracting archive. HPDIR.EXE and HPCOPY.EXE use BIOS calls to read from a PC 3.5" drive HP LIF floppies formatted using option 16 of MEDIAINIT. HPDIR displays the directory of an HP floppy, and HPCOPY copies one or all files from the HP floppy to a DOS directory. Programs are courtesy of Pier-Luigi Lollini, Instituto di Cancerologia, Universita' di Bologna.

Get Programs from CD-ROM
To unpack, simply copy the program to an empty directory and execute it.

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This is a pair of programs which transfer files, including listmode files, between Hewlett-Packard (HP) Pascal 3.1 computers and PC's. Transfer can occur in either direction. Hence, PC copies of files can serve as backup which can be transferred back to the HP for analysis with HP software. PC copies of listmode files can also be used with analysis software such as WinMDI and MFI.

Transfer takes about 30 seconds per 10,000 event listmode file. Alternative transfer software sold by Verity, which uses the same hardware, is said to transfer files considerably faster. (Verity Software House, Inc., Topsham ME USA. 207-729-6767.

Use of these programs requires an IEEE-488 GPIB card for the PC. Such cards cost about $500. One vendor which provides excellent technical support is National Instruments,, accessible at the National Instruments web site. Another solution is the OptiMATE package (board plus fast commercial software) from Applied Cytometry Systems.

The PC program HPPCLINK.EXE is included in the self-unzipping file HP2PC.EXE.

The HP program HPPCLINK.CODE can also be gotten here bundled with tools which enable you to put it on an HP-formatted diskette. The instructions, in HPPCLINK.TXT, are included in the bundled program, X2FF10LK.EXE. Thanks to Scott Rouse for figuring out how to do this! (This method will allow any public domain HP program to be transferred via Internet.)

For further guidance on four possible ways to transfer files between HP's and PC's, see the file HP2PC.TXT.

These programs were provided to the public domain by Becton-Dickinson.

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HP2PC (HP->PC via serial cable)

This is a pair of programs which transfer files, including listmode files, between Hewlett-Packard (HP) Pascal 3.1 computers and PC's via a serial port cable. Transfer is implemented only in the HP to PC direction. PC copies of listmode files can be used with analysis software such as WinMDI and MFI.

Transfer takes several minutes per listmode file. Thus, transferring a group of files can take several hours. To make matters worse, the HPTOIBM program contains a bug which makes it transmit only every second file. After it has been run, if the first file in the series is moved elsewhere, and the program restarted, it will transfer the alternate files it skipped during the first run. Thus, the program must be run twice for every group of files to be transferred. This bug was fixed, but the fixed version of the program has not been located in recent years. In particular, inquiries with Becton-Dickinson, and with the author, Morgan Conrad, have been fruitless.

The PC program FROMHP.COM is included in the self-unzipping file HP2PC.EXE. The HP program HPTOIBM.CODE must usually be obtained by snail mail on an HP-format diskette. This is because most HP Pascal 3.1 computers do not have an ethernet port, so the diskette drive is the only way to get an external file into the computer. Ethernet ports for these computers are so expensive as to be prohibitive for most facilities. To put it another way, if your HP had an ethernet port, you wouldn't need HPTOIBM.CODE! To obtain the HP program, contact

For further guidance on four possible ways to transfer files between HP's and PC's, see the file HP2PC.TXT.

These programs were provided to the public domain by Becton-Dickinson.

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Resonance Energy Transfer Intensity

Calculates RETI, e.g. for donor = fluorescein, acceptor = tetramethyl rhodamine. Requires input of 12 values (4 autofluorescence, 8 donor-excited [e.g. 488 nm; 4 detected green, 4 red], 4 acceptor-excited [e.g. 514 nm]) to calculate one RETI value. Math checked by Janos Szollosi. Input batched via ASCII file. MFI (see above) was designed to generate the 12 input medians efficiently. Author: Eric Martz, Univ. Mass., Amherst MA USA.

If interested, request by email to have the latest version put up on the ftp server.

Get RETI by from the CD-ROM.

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Corrected Fluorescence Intensity

Accepts log-acquired median or mean intensities typed manually. Converts log values to a linear intensity scale, optionally subtracting control value and/or rescaling to MESF. C source code included. Author: Eric Martz, Univ. Mass., Amherst MA USA.

Get CFI program from the CD-ROM.
CFI Readme file and License.

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Sort Recovery Calculator

This is a JavaScript program written by Joseph Trotter at the Scripps Institute, which calculates estimated recovery statistics for sorting.
Run the script.

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Problems getting program files?

All the DOS programs in this collection are in the form of ZIP archives or self-extracting EXE files.
There is a free version of UNZIP.EXE here.

Macintosh programs throughout the disk are in the form of BinHex encoded Stuffit Expander archives. Some Mac programs are self-extracting (after being un-BinHexed), while others must be explicitly extracted by a StuffitExpander compatible prlgram.

Stuffit Expander is available here, but it is in BinHex format also, and you must acquire a program capable of decoding BinHex documents elsewhere.

The browsers used to navigate this CD should offer the option of downloading these files to your hard disk when the links are clicked on. In addition, you can copy the files directly from the CD to your hard disk. Each of these programs is in its own directory in the \3\data\ directory of the CD.

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