Using the MAS on the FACS Calibur

Instrument setup using CellQuest.

Summary of differences between MPM and WorkList Manager.

Introduction

With the MAS, you can acquire your FACS data from cells in 96 well plate format. Samples can be in traditional round bottom or V-bottom plates, or in 1- or 2-ml deep well plates (although only the specific plates listed in Materials and Equipment can be used). This permits you to perform staining procedures in 96 well plates, including whole blood protocols, saving you significant time.

This document describes how to use the MAS, including materials, instrument setup, and using the MPM software.

Outline of an experiment using the MAS

Stain your cells in a plate compatible with the MAS. Include compensation samples. Make sure that the volume of the buffer used for the final resuspension of the cells does not vary significantly from well-to-well.
Transfer your compensation samples to FACS tubes (12 x 75 mm, from BD).
Before turning on the instrument, check the volume of fluid in the sheath cubtainer and the waste cubtainers. If appropriate, either replace the sheath cubtainer or replace or empty the waste cubtainer.
Turn on the components of the cytometer in the following order: (a) the FACSFlow supply, (b) the MultiWell Autosampler power, (c) the cytometer, and (d) the computer.
Take the outer metal tube of the droplet containment module (DCM) off the sample injection port (SIP).
Launch CellQuest and use it to obtain your compensation and PMT settings. Save the Instrument Settings in your own folder in the FACS Users folder. Unlike Worklist Manager, the settings file does not have to be stored in the "BD Files:Instrument Settings" folder.
In CellQuest, construct a CellQuest Template Document that you will use in the MPM software. The CellQuest Template will contain typical acquisition plots. You must set Acquisition Parameters to use "Events or Time", and the time must be the lesser of 250 seconds or the volume of your sample in microliters. The data in the Parameters Description box will not be used by the MPM software (nor are Panels used, in contrast to WorkList Manager). Save the CellQuest Template document in you own folder in the FACS Users folder.
Install the Cytometer Interface Unit (CIU) on the SIP.
Launch the MPM software.
Set the cytometer on "Run" and "High". Run the Prime System command from the MPM Autosampler menu two times.
Set up MPM parameters.
Run MPM.
When you are finished running your samples, with the CIU in place run the Prime from the Autosampler menu 3 times.
Remove the CIU from the SIP, replace the outer tube of the DCM, and manually wash the cytometer system for 5 minutes with 10% bleach, followed by 5 minutes with 10% water.
Transfer your data to an appropriate place.
Turn everything off.

Materials and Equipment

Plates

Only plates on the list specified in the document Plate_Definition_Guide.pdf may be used with the MAS. The MAS contains a sample probe that is programmed to descend into the wells of your sample plate. The depth that the probe descends is set by parameters that are associated with a particular plate. The plates that we are most likely to use are listed in the table below. Make sure that you have some of these on hand.

Multiwell Plate Definition	BD/Falcon Cat#	Description	Material	Bottom Type	BD VWR Cat#	Corning equivalent
351190	351190	96-well, storage plate, 340 µl volume	PP	U	12777-030	3365
355263	355263	96-well, storage	PP	V	BD353263	3363
353910	353910	96-well, clear	PS	U	62406-015	3370
353964	353964	96-well, deep well, 1 µl	PP	Conical	62406-494	3959
353966	353966	96-well, deep well, 2 µl	PP	Conical	62406-496	3961
353968	353968	96-well, deep well, 350 µl	PP	Conical	62406-510	-

Vortexer

When vortexing samples in deep well plates, it is necessary to use a vortex mixer that does not have significant up-and-down action. We prefer the Titer Plate Shaker Lab-Line (VWR catalog #57019-600).

Multichannel pipettors.

Alternatively, samples can be mixed by pipetting up and down. For the large volumes associated with work with whole blood, multichannel pipetters are available from Matrix Technologies that are programmable and that deliver up to 1250 µl, and are capable of mixing. See the following table for features.

Pipetter	Catalog #	Spacing	Channels	Volume	Programs	~Price
Impact	6004	Fixed	8	1250	1	$1099
Impact²	2004	Fixed	8	1250	6	$1319
Equalizer Impact	6034	Variable	8	1250	1	$1575
Equalizer Impact²	2034	Variable	8	1250	6	$1794

The catalog numbers for compatible pipette tips are found at the Matrix web site.

Bottle-top dispensers

If an 8-channel pipetter that delivers ≥ 1 ml is not available, it is possible to use a bottle-top dispenser. The dispenser must be used to deliver the liquid with sufficient force to achieve mixing, but gently enough to prevent contamination of samples between wells. The Bottle-Top Dispenser from Barnstead, or equivalent, may be used.

Fluidics system of the MAS

The following is from the BD pdf document BD Multiwell AutoSampler User's Guide.

The FACSFlow Supply System is an automated sheath and waste fluid control system designed for use with the BD Multiwell AutoSampler.

The FACSFlow Supply System includes:

FACSFlow Supply System unit with two removable bottom plates
modified four-liter sheath tank for the cytometer
modified four-liter waste tank for the cytometer
probe sets and tubing with color-coded connectors
probe holders
sheath and air filters

A complete description of the FACSFlow system can be found in the PDF document BD Multiwell AutoSampler User's Guide, present in the printed manual for the MAS, or available on the Mordred or FACS Lab computers.

The Cytometer Interface Unit (CIU) is installed in place of a FACS tube when performing acquisition from plates. Otherwise, conventional 12 x 75 tubes can be used. When not in use, the CIU is mounted on the FACS Calibur via magnets found on the top or the side of the CIU.

Before installing the CIU, it is necessary to remove the Droplet Containment Module (DCM) shield from the Sample Injection Port (SIP). Unscrew the fitting that holds the DCM shield onto the SIP, slide the DCM shield down off of the SIP, slide the screw fitting off of the DCM shield, slide it back onto the SIP, and rescrew it into place. Set the DCM shield in a safe place, as you must reinstall it when you are finished with the MAS. You will not need to manipulate any of the fluidics system behind the plate door of the MAS.

Setting Things up in CellQuest

Before running MPM, it is necessary to run CellQuest for the following reasons:

Obtaining instrument settings, which may be stored in your own user directory.
Note that this is different from the situation with Worklist Manager, where the instrument settings must be stored in the BD Files directory.
Setting up and saving a CellQuest template, which includes appropriate acquisition dot plots, gates, and statistics panels. This template can be saved anywhere as well, though I recommend that you save it in your own user directory.
Setting up "acquisition parameters" specifying that the collection criteria must be set to "Number of Events or Time", and the time must be set to less than 250 seconds. The number of events can be any number, and depends upon the frequency of your target population. This is automatically saved with the CellQuest template.

Compensation control samples are set up in the usual way. If you have stained all of your cells in a plate, you probably want to transfer the compensation control samples to 12x75 FACS tubes before running them manually in CellQuest. I had one technician figure out how to run compensation samples from within MPM, but it's tricky and I'm not sure I recommend it.

MPM Software

The MAS is controlled by the MPM software. As described below, MPM works with CellQuest in a way that is similar—but not identical—to the way that Worklist Manager works with CellQuest.

Setting the User

When you open the MPM software, you are asked to select a user from a pull-down menu. You can select "General User", or set up a User for your self. The General User has access to the keyword subsets and protocols established by all users, but an individual user can select from among only keyword subsets and protocols that were established by that individual user.

Priming the system

Before running the MAS system, it is necessary to prime the fluidics two-to-three times. You might as well do it at the very beginning.

Setting initial and final washes

The initial and final washes flush the inside and outside of the probe, CIU, and CIU tubing. You can set the MPM software to perform 0-4 washes. Use at least two. To set up washes, select the command:

Autosampler > Set Initial and Final Washes

Plate configuration

The MAS system can acquire samples from a variety of different plates, as noted in the Plate Definition Guide. Since these plates have different well depths, it is necessary to tell the MPM software which plate you are using. Choose Layout > Plate Configuration, and select your plate from the pull-down menu.

Software settings

The following sections describe important software settings within MPM.

Filenames

The filenames for your FCS files will all have the same prefix (in contrast to filenames that are used in Worklist Manager, which have prefixes that vary from sample to sample within a panel). There are three fields in which you can enter parts of the prefix. I suggest that you separate the parts of the prefix with underscores ("_"). For part I of the prefix, I suggest that you use your initials, followed by the date, in the following format: "JDA_020627_". The second and third parts of the prefix can be used to indicate specific information about the experiment, as in the following examples:

Example Number	Part II	Part II Explanation	Part III	Part III Explanation
1	RhM2_Gr1_	Rhesus experiment M2, Group 1	Week54	Week 54 of the experiment
2	HuTetScreen_	Tetramer screen of a new donor		This part might be blank
3	LCMV_IFNg_	IFNγ stimulation experiment in an LCMV-infected mouse	Balbc	This experiment was done in Balb/c mice.

You don't need to use an underscore following the last of the prefixes that you use; the MPM software will add a "." followed by the well designation.

The filenames in MPM will sometimes contain less information than filenames established by Worklist Manager. For example, if you are analyzing samples from many donors that were processed on a single plate, it will not be possible to indicate the donor numbers in the filenames. However, as we'll see below, this can be done within keywords that are contained within the FCS file.

Instrument Settings

Instrument settings files are established from within CellQuest. In contrast to the situation with Worklist Manager, they can be stored in any folder. I suggest that you store them in a folder that you create for yourself under "FACS Users".

CellQuest Template

Like Worklist Manager, MPM requires you to establish a Template in CellQuest. The CellQuest Template must be set up to contain:

Acquisition dot plots and histograms
Any statistics you wish to display during acquisition
Acquisition parameters.

In the acquisition parameters panel, it is necessary to set to acquire "Number of Events or Time", and the time should be set to 250 seconds or less. However, unlike the situation with Worklist Manager, the panel settings in the Parameter Descriptions are not used by MPM. Consequently, the labels for your parameters are not displayed during acquisition, but they are contained in the FCS files saved by MPM.

Data Storage Folder

It is necessary to tell MPM where to store your FCS files. Do this by hitting the Data Storage button in the Setup tab, then navigate to the folder you would like to store you data in.

Keywords: A Crucial Means for Annotating Your Data

Since the file names created by MPM for a single experiment all contain the same prefix, it is more crucial than ever to use Keywords to properly annotate your data. You can use Keywords to indicate properties such as donor numbers or names, mouse strains, sample tissues, staining methods, time points in an experiment, etc. In fact, the Keywords option in MPM is extremely flexible, although how to set them up and use them is not obvious at the outset.

Defining a new Keyword

You can define a keyword such as "Sample Type: Human". You can associate with each keyword a list of options that can be selected from a pulldown menu. For example, members of the "Sample Type: Human" list might include: (1) WB: EDTA, (2) WB:Heparin, (3) WB:ACD, (4) PBMC: Fresh, (5) PBMC: Frozen, and (6) Lymph node. Another user might have set up a different keyword called "Sample Type: Mouse" that includes: (1) Splenocytes, (2) PBMC, (3) Thymocytes, and (4) Lymph node. Either use might have set up a keyword such as "Staining Protocol" that might include: (1) Surface, (2) ICC: Pharmingen, and (3) ICC: Becton Dickinson.

Defining Keyword Subsets

In the paragraph above, it was obvious that users performing experiments in mice and in humans might wish to use different keyword subsets. These can be selected in....

Defining stain names

To be completed

Acquisition parameters used in MPM

Wash and mixing cycles

The wash cycle is used to minimize carryover from one sample to another. The default value is for a single cycle only. It remains to be determined if additional cycles are required. The MAS unit will mix the samples in each well by pipetting up and down. The default value is for a single cycle. It remains to be determined if additional cycles are required.

Sample volume

The Sample Volume parameter instructs the syringe pump on the MAS to pull up the specified volume. In order to avoid introducing bubbles into the flow cell, this should normally be at least 30 µl less than the volume contained in the sample wells. For example, if your sample wells contain 200 µl, then the largest value that you should use for Sample Volume is 170 µl.

The maximum sample volume that can be used is 250 µl.

The dead volume of the system is approximately 30 µl. This means that if your sample volume is set to 250 µl, you should expect to acquire data for only 220 µl, or 12% of your sample. This loss cannot be avoided.

Stuff below here to be edited

Steps for setting up and acquiring data.

In CellQuest, obtain file settings as usual.
In CellQuest, set up an "Acquisition Template". This template contains plots, stats, and acquisition parameters. For the acquisition parameters, the collection criteria must be set to "Number of Events or Time", and the time must be set to less than 250 seconds. The number of events can be any number, and depends upon the frequency of your target population.
The Instrument Settings file and the acquisition template can be stored anywhere. I suggest that you store them in your own subfolder under " FACS Users", rather than in a shared folder.
Quit CellQuest
Open the MPM software from the folder "BD Applications" in the Apple Menu.
Select the "Acquisition" button from the first dialog box.
Select yourself from the "User" pulldown menu in the second dialog box, or click on the "New User" button if this is you first time using MPM.
The MPM software takes some time to boot up. Be patient
When the MPM window first appears, select the "AutoSampler Parameters View" tab if is not selected.
Set the file name prefixes. You don't need to use all three. I suggest that you use an underscore after all but the last of the prefixes. The same prefixes will be used for all of your samples.
Click the "Acquisition Doc" button and navigate through the file system to find your acquisition template. Note that MPM does not appear to use standard Macintosh dialog boxes.
Click the "Instr. Sett. File" button and navigate through the file system to find your file settings.
Click in the "Sample Vol" field and enter a number that is ≤ 250. Note that you should have at least 30 µl more volume in your sample wells than the number you enter here.
Click in the "Mixing Vol" field, and enter a mixing volume that is approximately 1/2 of the volume in your wells.
Click on the "Data Storage Folder" button at the upper right and navigate through the file system till you decide where you want to store your data. You can make new folders in this dialog box.
From the "Current Plate" pull down menu, select you current plate. The plate numbers are described in the document "Plate_Definition_Guide.pdf" that is available on the Mordred or FACS-G4 computers. It is very important that you use only those plates that are described in bold in this document, and that you select the appropriate plate from the pull-down menu. This parameter determines the depth that is used by the sample probe.
Click on the "Keyword Subsets" tab.
To set up new keyword subsets, select "Modify > Modify Master Keyword Set" from the "Layout" menu. This will open a spreadsheet like dialog box. Click the "Add" button to add new keyword fields. Click in the Keyword cell to change the name of the keyword. Click on the "Edit Choices" button to construct a list of keywords that can be selected from a pull-down menu. Click on the "Choose From List" check box to force choices from the menu.
When you have set up as many keyword fields as are required for your experiment, select the rows that you want to group together in a "Keyword Subset". For example, you might have keywords such as "Sample Type", "Donor Number", "Lysing Protocol", and "Mouse Strain". For human experiments, you might wish to group the first three together in a subset called "Human". After selecting the rows, click on the "Define Keyword Subset" button, and enter the name of the new subset in the "New Entry:" field. Click on the OK button to leave this dialog, then on the "OK" button again to leave the "Master Keywords Set" dialog box.
Select the cells that you'd like to describe with a particular keyword. You can select the whole plate by clicking in the upper left hand corner of the plate, and you can select rows or columns by clicking on the row or column headings. You can select single cells by clicking in them, you multiple continguous wells by dragging across a number of wells, or multiple discontinuous wells by holding down the "Control" button and clicking them (the programmer was obviously not a Mac person!).
For now, ignore the "Panels" and "Overlays" sections. Panels doesn't work, and I don't know how to use "Overlays".
Click on the "Protocol Parameters View" tab. This is where you'll enter you reagents for you fluorescent channels.
If all of your positions on your plate contain different stains, these can be entered cell by cell. Otherwise, follow the instructions below to reduce you typing.
Select the wells for the common reagents first. For example, if you are using CD3 FITC and CD8 PerCP for every sample, click on the corner of the plate layout to select all of the wells, and then enter CD3 FITC in the FL1 field and CD8 PerCP in the FL3 field.
Next select wells for members of a panel. For example if every sample in column 1 uses CD94 PE, then select column 1 and enter CD94 PE. Do the same thing for the rest of the columns, and then for the rows.
You're almost ready to start your run. There is one thing you need to do first, however.
PRIME THE SYSTEM! This is distinct from the "Prime" button on the front of the cytometers. The Prime System command is available from the "Autosampler" menu. Do this at least once, if not twice, before you start your run.
Select the wells you'd like to run. This is distinct from the keyword and protocol parameters assignments you've set up. In addition, you can select the wells in any order, and they will run in the order you've selected. Once you've select the wells, do not click on the plate again.
Save your protocol. Select the "Save Protocol" command from the Save menu. Protocols are stored in a "HD Name > BD Applications > MPM > Users > UserName" folder.
Start your run. To do so, select "Acquire" from the Acquisition menu. Then, click on the "Acquire" button.

Shut down

Run a 1/2 tube full of bleach for 5 minutes.
Run a 1/2 tube full of water for 5 minutes.
Shut down the computer, cytometers, and FACSFlow supply system.

Trouble shooting

If you need to pause you run, I don't know how the system handles this if done from within CellQuest or MPM.
I do know that you can hit "Pause" in MPM, and then skip defined wells (for example, if you have a clog, etc.

Created on 6/9/02 12:56 PM 1 Modified: 8/31/03