Cyan

Using the CyAn

Users may be trained to run the CyAn themselves or may elect to have Facility staff run the samples. It is cheaper per hour to run the CyAn yourself. However, usually, unless you are doing flow regularly and in sufficient quantity, it is better to have us run the samples. You are likely to get better data and get it sooner than running it only occasionally yourself. If you anticipate doing a substantial amount of flow then you should be trained. We offer training courses regularly and would be glad to teach you. If you run it yourself, the brief instructions below will help remind you of the major important points in operating the instrument.

Prior to your training, we recommend that you view the Beckman tutorials on Introduction to the CyAn, Operation of the CyAn and Summit Software: http://www.coulterflow.com/bciflow/theory.php

The figure below describes the basic filter configuration of the CyAn. All three of our CyAns are identical. They each have a 25mW Coherent Sapphire 488nm (blue) DPSS/OPSL laser, a Coherent 100mW 405nm (violet) laser, and an 60mW Laser Concepts red diode laser. Please - if you do not see the fluorochrome you wish to use shown below, do not use it without first talking with us to determine if we can accommodate it. The fluorochromes preceded/followed by an * are fluorochromes we have filter sets already available for.

Additional fluorochromes may be accommodated but will likely require us to obtain NI and/or dichroic filters - users are expected to provide 50% of the cost of these filters. Please consult the Facility Director to discuss your needs.

 

 

CyAn Quick Startup Instructions

1) If the CyAn fluidics cart and lasers are on, skip step 4.   
2) Log onto the computer using the AD domain - Use your ONYEN and associated ONYEN password - If the computer should be off turn it on first.
3) Start Summit (it should be on the desktop) - If it is not, find the Summit icon on the C: drive and locate the Summit 4.3 icon.  "Send" it (i.e. make a shortcut on the desktop).  PLEASE DO NOT delete the icon or drag the icon to your desktop, as it will be unavailable to other users - Select a database (use the browse button) or create a new database. Databases can be saved on the D: drive in a folder that you may create- you can name the folder after your flow facility assigned UserID , which is assigned after the hands-on CyAn training. Use a logical name for your database name - Most users will need only 1 database. Click on OK and Summit will finish loading.
4) Open Instrument Control Panel - Click on "Startup" button - follow directions on screen on upper left corner of main display screen - wait at least 30 minutes for lasers to warm up.
5) Complete Acquisition Control Panel fields:
- Define Sample and File naming formats
- Create new database samples folder
- Define path to save data - create new data folder
- Set sample number to "1" if desired
- Define limit type and amount if desired
6) Select parameter to use for trigger:
Usually the channel for DNA content in DNA analyses
Usually FSC for others like immunofluorescence
7) Name parameters and select data types (Height (peak), Area or Log) to be used for each parameter - remember width is always collected but only for the trigger parameter
8) Create histograms using Histogram Control Panel
Create a single parameter histogram by double clicking desired parameter name under X-column
Create a two parameter histogram - one parameter for X-column and one from Y-column
9) Check that boost and auto-backflush are set to what you prefer (Edit Preferences/Instrument/CyAn)
10) Clean the sample probe using DI water - select “sample clean” from Instrument Control Panel
11) Put on a negative control sample
12) If auto-boost is set to off use manual boost on Instrument Control Panel to start sample. Clear data after boost finishes. Adjust sample pressure as necessary If you don't see any data during the boost either you don't have any cells or the CyAn is clogged. Request staff help.
13) Adjust FSC gain - Adjust PMT voltages (linear or log signals) or PMT gain (linear signals only). Place cells on FSC/SSC plot so all desired cells are on scale. Adjust PMT voltages on log fluorescence channels so negative cells are at least centered geometrically in the first decade.
14) Set up an event filter (select “Acquisition” from main menu bar, then “event filter”) If dialog does not open, histograms are populated with saved data. Collected a tiny data set and do not save then attempt to set up event filter.
15) If needing compensation run single color controls and save data files.
16) Set up auto-compensation (menu icon on bottom most panel of the "Sample Control Panel".
Run Samples
17) When finished clean instrument - follow protocol below.

Cleaning/Shutdown

We have found that the cleaning procedure for the CyAns needs to be altered.  Effective immediately (May 2011)  please follow the procedures below completely.  These will also be posted at the machines.  It is important to follow these procedures exactly to reduce problems (clogging primarily) with the instrument and to avoid downtime.  Remember - the machine is to be turned off when you finish if the next person is scheduled for 4:30pm or later and you do not know in some positive way that the next person will actually use the machine.

Note:  All users, regardless of application, should follow the new procedure below.  Fill up all tubes to and only to the level of the BOTTOM of the color coded tape.

Cleaning procedure when you are leaving the instrument on for the next user:

1.      Using the larger (17mm diameter) cutoff tube that is color coded red, fill the tube to the bottom of the red tape using the bleach squirt bottle.

2.      Place the tube on the machine and close the lever – you will need to hold the tube until the lever is under it.  If “Auto-backflush” is turned off, you will need to click the “Backflush” button on the instrument control panel.  Allow the backflush to complete.

3.      With no tube on the sample probe, perform a backflush and rinse the probe using the distilled water squirt bottle.

4.      Repeat steps 1 and 2 with Contrad using the larger diameter cutoff tube labeled with green tape (use the Contrad squirt bottle to fill the tube).

5.      With no tube on the sample probe, perform a backflush and rinse the probe using the distilled water squirt bottle.

6.      Repeat steps 1 and 2 with distilled water using the larger diameter cutoff tube labeled with blue tape (use the distilled water squirt bottle to fill the tube).

7.      With no tube on the sample probe, perform a backflush and rinse the probe using the distilled water squirt bottle.

8.      Click on “Sample Clean” on the instrument control panel.

9.      Using the 12×75mm tubes, first select the bleach tube (color coded red) and fill with bleach to the bottom of the color coded tape using the bleach squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 1 minute.

10.  Remove tube and backflush while rinsing the probe with distilled water.

11.  Click on “Sample Clean” on the instrument control panel.

12.  Using the 12×75mm tubes, select the Contrad tube (color coded green) and fill with Contrad to the bottom of the color coded tape using the Contrad squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 1 minute.

13.  Remove tube and backflush while rinsing the probe with distilled water.

14.  Using the 12×75mm tubes, select the distilled water tube (color coded blue) and fill with distilled water to the bottom of the color coded tape using the distilled water squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 1 minute.

15.  Remove tube and backflush.

16.  Place the 12×75mm tube of distilled water back on the probe – DO NOT close the sample lever.

17.  Close Summit and log off the computer.

Cleaning procedure when you are shutting down the instrument:

1.      Perform steps 1-7.

    2.      Click on “Sample Clean” on the instrument control panel.

    3.      Using the 12×75mm tubes, first select the bleach tube (color coded red) and fill with bleach to the bottom of the color coded tape using the bleach squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 1 minute.

    4.      Remove tube and backflush while rinsing the probe with distilled water.

    5.      Click “Shutdown” button on the instrument control panel.

      6.      Using the 12×75mm tubes, select the Contrad tube (color coded green) and fill with Contrad to the bottom of the color coded tape using the Contrad squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 1 minute.

      7.      Remove tube and backflush while rinsing the probe with distilled water.

      8.      Using the 12×75mm tubes, select the distilled water tube (color coded blue) and fill with distilled water to the bottom of the color coded tape using the distilled water squirt bottle.  Install tube on sample probe and close the lever – cleaning will run for 30 seconds.

      9.      When the distilled water finishes running the computer will shutdown the lasers and fluidics.

      10.     Be sure to leave the tube of distilled water on the sample probe.

      11.     Close Summit and log off the computer.