Coulter Multisizer 3 OperationFebruary 12, 2007
TRPL Doc. D210
By: Angel Varela-Rohena
Modified: Richard Carroll
Page 1 of 6
Coulter Multisizer 3Operation
Introduction:
The Coulter Multisizer 3 (MS3)enables accurate quantitation of cell populations, as well as measurement of other important cellular parameters, such as cell volume. In practice, there are several factors that must be considered when using the MS3. Failure to consider these factors could lead to generation of inaccurate and misleading data. First and foremost, it is essential that each user have his or her own SOP and SOM. SOP and SOM generation is described in parts A and B of Procedures (below). Secondly, the Multisizer cannot distinguish live cells from dead cells or large cellular debris. So if your population contains a lot of debris and or dead cells, cell counts should be verified on a hemocytometer using trypan blue (see Document D480).
Procedures:
A.Mapping Directory to Save on the Network
1.Log on computer:
a.username: coultercounter
b.password: c0ult3r (this is a zero, not an “o”)
2.Go to Start, then “My Computer”
3.Click on the Coulter Counter Data network drive (Coulter Counter data on ‘Abrmsns0’, (Z:)).
4.Create a folder with your nameunder the MS3 folder that you are working on (either Room 582, or 543 or 577).
5.Close the window.
6.Open Multisizer 3 software from the desktop shortcut.
7.Click on Sample Information: Fill up as shown in Figure 1.
Figure 1. Sample Information Page
B.Setting upanSOP:
1.If not already open, open MS3 software from desktop shortcut.
2. In the window on the left, under SOM, click “change”
a.Click “Directory”
b.Click “Browse”
c.Go to “Coulter Counter Data”
i.Pick the appropriate room (MS3 577, 543, or 582)
d.Choose your username folder.
e.Hit “OK”
3. In the SOM window:
a.Make sure that volumetric is set to 500 uL and number of runs is 1.
b.Under “After each run”, check on “save file” and “include pulse data”.
c.Click “file name” and check the following three boxes:
i.Sample ID (25 characters)
ii.Date
iii.Unique Number (3 digits)
d.Then click “Use Example” and hit “OK” to close.
e.On “Aperture”,make sure it says 70 um.
f.OnThreshold, make sure “Sizing Threshold” is set to 1.44 fL or 1.46fL (depending on instrument).
i.If not, click on “use default”, then Hit OK.
g.Hit OK to close SOM dialog.
4.Go to “Settings” pull-down menu.
a.Click Under “Convert Pulses to Size.”
b.Set “Size Bins” to 300
c.Under “Bin Spacing”, select Log Volume.
d.Set the gate to 30-4000 fL.
e. Hit OK
f.While under “Settings”, click “Save SOM”.
i.Click on “SOP” folder and save under “SOM” folder.
5. Go to“Preferences”
a.Click on “printed report”
i.check on “Sample info” and “Short”
ii.On Size, check “Graph”and “Statistics”
iii.On Size statistics, check mL(the first one), then hit OK.
b.While still under “Preferences”, go to “Graph Options”.
i.Select Outline for graph style and hit OK.
c.While still under “Preferences” click on “Save Preferences as”
i.Select “SOP” folder
ii.Open “Preferences” folder
iii.Save preferences here
6. Create the Actual SOP
a.Under settings, click “Create an SOP.”
b.Navigate to the appropriate SOM and Preference filescreated above.
c.Save your SOP in the SOP Folder.
C.Sample Preparation:
1.Using the dispenser, place 20 mL of Isoton II buffer in an accuvette vial.
a.Make sure that the full 20 ml have been delivered.
2.To the vial, add 40 ul of cell suspension.
D.Sample Running:
1.Open “Multisizer 3” from the desktop.
2.In the “SOM” panel, make sure to select your file directory on Coulter Counter Data on the “Z” drive.
3.Go to Settings and click on “Load SOP”
4.Navigate to the SOP folder and select your SOP from the directory
5.Carefully remove blank vial by squeezing the front of the stage and lowering it.
6.Invert sample twice with the cap firmly on, remove cap, and put sample on stage. 7. Squeeze the stage in order to gently lift it and dip the probe into the vial.
a.Take extreme care not to damage the probe by banging it into the bottom or side of the vial.
8.In the “Sample Info” panel, click change, and enter the following:
a.On Group ID, enter the date.
b.On operator, make sure the initials are yours
c.Change the other fields as needed, and then hit “OK”.
9.Click “Start” to acquire your sample.
10.Monitor your sample during the run
a.run time should be ~ 27.5 seconds.
i.otherwise, the counter may be clogged (see Step F1, Clog Prevention and Removal, below).
b.Also observe the “concentration” monitor and make sure that it is not in the red more than transiently. Excessive time in the red also indicates a clog.
11.When the run is finished, proceed to analyze the sample.
12.To gate on a particular population, follow one of the two procedures described in this step or Step 13:
a.create cursors with the mouse
b.go to “analyze”
c.Click “Convert Pulses to Cursors” to zoom on the population of interest.
13.Alternatively, go to “Ana;yze”
a.Select “Convert Pulses to Size” if you would like to change the window size to a particular range (e.g. 100-1500 fL for activated T-cells).
14.To print analysis, go to “RunFile”, click on “Print Report”.
15.Hit “Reset” to clear window and to ready the machine for the next sample.
16.Remove sample, pour liquid into waste container and discard counting vial in biohazard trash.
17.Count another sample if needed. Remember to change the “Sample Information” for each sample.
18.When done counting samples, remove last sample, discard it and replace with a blank vial containing Isoton II.
E.Post-Sample Running:
1.Under Settings, click on “Remove SOP”.
2.Under SOM, change directory back to “C:\ MS\Samples”.
3.Under System, click “Flush Aperture Tube”.
4.Hit the “empty waste tank” button (see Figure 2).
Figure 2. Waste Tank Button.
F.Miscellaneous:
1.Clog Prevention and Removal:
a.To prevent clogs, it’s recommended to rinse the counting vials with clean water before pouring Isoton II.
b.During acquisition, monitor the probe thru the small window on the left side to detect large particulates or cell clumps that might block the probe. Adjust the focus with the optics alignment knobs (See Figure 3).
Figure 3. Optics Alignment Knobs.
c.If you or the software detect a clog:
i.Go to “System”
ii.Select “Unblock”
iii.Monitor the progress through the small window.
d.If the unblocking procedures in part C do not work:
i.using gloved hands, remove the sample and gently rub the probe once with one finger.
ii.Lift stage to make sure probe is cleared.
iii.Go to “System”.
iv.Hit “Flush Aperture Tube”.
v.Re-acquire sample.
2.Waste Tank Changing
a.Gently unscrew the waste bottle lid and pour contents in the sink.
b.Add a small amount (~ 100 ml) of bleach to the bottle.
c.Screw the lid back on.
d.Gently tap the bottle to make sure the sensor is free.
e.At the same time the waste bottle is changed, fill up the Isoton bottle as well.