Flow Cytometry

Starting the Guava (BG HT)

• Turn on the Guava (located in S305) by pressing the horizontal switch located on the back right of machine

1. The light on the front of the machine will light up when it is ready

• On the right side of the machine are a waste vial (red label) and a rinse vial (blue label)

1. Unscrew each vial by twisting the glass while holding caps stationary to avoid harming the tubing
2. Dispose of anything in the waste vial as necessary
3. Fill the rinse vial with MilliQ water
4. Reattach the caps to the vials

• On the computer, open software “guavaSoft 3.3”

1. Located on the desktop, shortcut image is a big “M”
2. Software will open to its main menu

Cleaning the Guava (BG HT)

If machine is already clean, skip this section

• Click “cleaning”
• Click “start clean”

1. Drawer will open
2. Check charts posted to the left of the Guava for tube placement. Make sure the water and ICF tubes are full and waste tubes are empty. There is MilliQ water in the squeeze bottle next to the Guava. There are instrument cleaning fluid (ICF) in the green capped bottle next to the Guava

• Look at the shelf from the side to make sure all tubes are level

1. This will help us avoid breaking the capillary

• In the dialog box, click on tube 9 to indicate the capillary shutdown position
• Click “OK”

1. Drawer will close by itself

• Wait for cleaning to complete

1. Will take ~17 minutes
2. While clean is running you cannot do anything with guavaSoft

• After cleaning completes (The bottom should say “Ready to Start”, click “Main Menu” at the top of the window to return to the main menu

Running samples

• Click “InCyte”

1. This will open a new window

• To begin acquiring data, click “Edit worklist”

1. Here you can open a previously made worklist (Using the “open” button on the right) or adjust the worklist settings
2. You can open a previous worklist or make a custom worklist by manipulating the options on the left.
3. The settings are listed below:

General

Acquisition order: Determines the direction the Guava will sample

1. “By Row” is recommended

Mixing Required: Check this box if you would like the Guava to use its metal paddle to mix each sample before acquisition

1. Mixing will take time and might not be recommended for fragile samples
2. Before Each Sample Wash: Determines the tools that the Guava will rinse before acquiring each sample
3. If not using the mixer, you should put “capillary only”
4. If using the mixer, you should put “capillary & mixer” unless mixing samples with the same paddle will not contaminate your samples

Cleaning Option: Indicates the cleaning method

1. “Clean and Rinse” is recommended for a 2.5 minute quick clean
2. Do Clean: Indicates when the selected cleaning method will occur
3. “Once at the end of plate” is sufficient, unless you have a sticky sample and would like to clean the machine more frequently

High Concentration Warning Limit (cells/µL): Determines the sample concentration where the Guava will warn you that the machine may not be able to distinguish between cells

1. Leave at “500”

Park Capillary In: Indicates where the machine will place the capillary after the run is complete to prevent it from drying out.

1. Set as “T09” to park capillary in position 9

Sample Specific

Acquire this sample: Allows you to set the locations where the machine will sample once you run the worklist

1. To acquire a sample, you should highlight the desired well or tube in the layout to the right and check the box
2. Samples that will be acquired will be shaded blue
3. You can deselect samples by highlighting the unwanted wells and unchecking the box

Sample ID: What each sample/well will be named

1. You can use this to label each sample if you want to spend the time on it
2. System will default the Sample ID to the location of the sample (i.e. “A01”, or “F11)

Events to Acquire: This is the target number of particles that the Guava will count per sample. 5000 events is usually sufficient

Time to Acquire: The amount of time the Guava will spend on each sample

1. The Guava will acquire a single sample until it reaches the designated number of events or until time runs out
2. Longer acquire times will allow you to get more events if the samples are very dilute, but will greatly extend the runtime of your worklist. 180-300 seconds/sample will usually be sufficient

Dilution factor: The Guava can only successfully read samples that are between 103 and mid-105 cells/mL, thus samples may need to be diluted to fall somewhere within this range. By putting in the factor by which you have diluted the sample (2, 20, etc.), InCyte can calculate the “full-strength” concentration of the sample for you. It is usually easier to just calculate this yourself, so this can usually be ignored

Original volume: Here you can put how much of the original sample you are using for this measurement. You can usually ignore this field as well Autosave FCS 2.0: By checking this box, the program will save the file as an older version of FCS file

1. If left unchecked, the program will save the file as FCS 3.0 by default (We want FCS 3.0, so do not check the box)

Replicate: This option will cause the Guava to acquire technical replicates from the same well

1. This will take more volume from the well, you will want to make sure that the sample is concentrated enough that the total volume taken does not drop the well volume below 50 µL

• Customized worklist settings can be saved for future use using the “save” button to the right
• Once ready, click “Run Worklist” to begin
• Determine the file name and location using “Select Output FCS” (We want to save under the Eric Miller folder)
• If a method and setting file already exists (Eric Miller Folder)that would be appropriate for your samples, choose the desired files for “Method” and “Settings”

1. You will still have a chance to adjust setting later on if desired
2. Leave the “compensation” section blank
3. If you would like to test or change the settings before running the whole worklist, press “Adjust Settings”
4. If you do not have a premade method or setting file, leave the fields for “Method” and “Settings” blank and just hit “Adjust Settings” to begin (See how to adjust settings below)

• Drawer will open

1. DO NOT put 96 well plate yet
2. Refer to the acquisition layout below for tube placement (or check "Sample acquisition setup” chart posted to the left of the Guava). Make sure the water and ICF tubes are full and waste tubes are empty. There is MilliQ water in the squeeze bottle next to the Guava. There is instrument cleaning fluid (ICF) in the green capped bottle next to the Guava.

• Ensure that all tubes are level by looking at the tray from the side
• Place 96 well plate in tray

1. Be sure to match A1 on plate with A1 on tray. MAKE SURE LID OF 96-WELL PLATE IS OFF
2. Make sure the plate is level by jiggling it within the tray and ensuring that it is not resting on the small ledge within the tray. An unlevel plate may lead to a broken capillary

• Once ready, click “OK” to begin
• Drawer will close, and plate will load

Adjusting Settings

• Only need to do this if you had to press “Adjust Settings” to change or create a new method
• Select desired well to test

1. Can be one of the selected wells or any other well on the plate if it contains a sample
2. Click on an appropriate blank well (negative control/media) to begin sampling from this well

• Set flow rate to “very low” to make sure you are not taking up too much sample while you are adjusting general settings
• Modify the number of plots you are interested in viewing using the “Plot” tab at the top of the screen

1. You can adjust the plots using the “Plot Type” menu at the top right corner of each plot
2. Adjust the axes of these plots by clicking on the axes labels and selecting the desired parameter (logarithmic “log” scale is recommended)

• If you set threshold to 0, you will still see particles showing up in the blank sample

1. Adjust the gain of each parameter to shift these particles toward the bottom left corner of each plot
2. Do not shift the particles so much that they are piling up on one of the axes
3. When you think you have adjusted the gains sufficiently, switch flow rate to medium and adjust gains as needed

• When you are ready to change the well that is being sampled, click the green arrow (next step)
• Click the icon located furthest left that looks like a blue circle (adjust settings)
• On the pop-up window, choose a new well
• Adjust laser gains and thresholds as necessary
• If a counting gate is desired, you may create a region for desired counting gate while adjusting settings

1. A count gate allows the machine to only count particles falling within a certain region and ignore the noise

• Then create a new gate that is defined by that region (click “tools”, “show gate list”, then in gate list window, name your gate, define the gate by the region name

1. The text of the “definition” HAS TO MATCH the name of the gate identically
2. If want events in gate to be colored the check “ColorGate” box
3. You can change the color by clicking the color in the box

13. Click the drop-down arrow for the “count gate”, click the gate that you want to count off

1. The default is Ungated/all events

14. Once everything is set, click the “next step” arrow (green arrow) 15. Click the floppy disk icon to save these settings for future use

1. This button is just to the right of “adjust settings”

16. Clicking the “next step” arrow again will initiate the run

1. The Guava is then fully automated and will complete the worklist
2. You may click the green “next step” arrow anytime it is lit to skip a step in the worklist as desired
3. You may pause (“pause” icon) or abort (“X” icon) the run at any time
4. If you abort the run, no file will be saved, and any unsaved settings will be lost