A Template defines sample-specific settings during tuning and acquisition. A Template contains:
• Introduction system settings
• Mass channels for event detection and monitoring
• Bead detection settings
• Acquisition system settings
• File types you want to acquire
Templates are shared across CyTOF® accounts. To see all Templates, open Template Editor (on the Instrument Control tab, click Template Editor).
A locked default Template for tuning—Default Bead Tuning Template—is applied to each Tuning Protocol and is set as the default template for manual tuning. This Template is intended for daily tuning and must be used by CyTOF Users. CyTOF Administrators can change the tuning template in custom Tuning Protocols for investigative purposes and when instructed by Fluidigm Service or Technical Support for troubleshooting.
A locked default Template for acquisition—Default Acquisition Template—is provided with the software. It includes:
• All EQ6 and EQ4 bead channels
• Cell identifier channels, such as iridium, rhodium, and platinum
• Monitoring channels
• Barcoding channels
Duplicate this template to customize it or create a new template.
A template must be applied to each sample in an Acquisition Protocol before you can start acquisition. The same Template does not have to be applied to each sample.
Bead channels and monitoring channels are always acquired for normalization and monitoring purposes, even if they are not included in the settings template.
The channels acquired depend on the selected bead type.
| Bead Type | Bead Channels | Monitoring Channels |
| EQ6 | 89Y 115In 140Ce 159Tb 175Lu 209Bi |
113In 142Ce 156Gd 160Gd 174Hf 176Lu |
| EQ4
(and EQ4-MDIPA) |
140Ce 151Eu 153Eu 165Ho 175Lu |
142Ce 156Gd 164Er 166Er 176Lu |
The Maxpar® Direct™ Immune Profiling Assay™ template (.tem) can be imported into Template Editor and used to acquire samples prepared with a Maxpar Direct Immune Profiling Assay kit.
For more information about Templates, see Create a Template.
Setting |
Description |
Introduction Settings |
|
Mixing Volume (μL) |
The volume of sample repeatedly aspirated and dispensed back into the tube during mixing.
|
Mixing Force |
The relative velocity of the sample when dispensed into the tube during mixing. The default setting is 10.
|
Stirring Speed |
The speed at which the probe stirs the sample. The default setting is 5.
|
Recover Sample |
When selected, any sample remaining in the sample loop is returned to the sample tube (with ~30 μL of CAS) when acquisition completes or stops. If Recover Sample is not selected, any remaining sample is sent to waste.
|
Sample Loop Agitation |
Check to agitate the sample loop for 400 ms every 2 sec during injection and washing. This setting is recommended to prevent cells from settling in the sample loop during acquisition and to reduce the risk of clogs.
|
Elements |
|
Element Table |
Click to view the Element Table. Click elements you want to add to the template, then click Apply.
(Optional) Add individual elements by selecting
the element from the drop-down list, then click
|
Mass, Element, and Label columns |
These fields are populated based on elements added to the list.
|
Target |
Enter the name or abbreviation for the biological target. The Target value appends the Label.
|
Event Detection |
When checked, the element is used for event detection. Unchecked elements are collected but not used for event detection. Elements are automatically selected for event detection when they are added to the list. |
Lock |
(CyTOF Administrators) To lock specific channels and labels within a template, check Lock. Locked channels can be modified by other CyTOF Administrators, but not CyTOF Users. Click the top-level checkbox to lock all channels in the template. |
Beads Detection |
|
Type |
Choose EQ6 or EQ4 depending on which bead type was used as an internal standard for your sample. The list of bead elements is displayed depending on which type is chosen. IMPORTANT Always choose EQ4-MDIPA for samples prepared with the Maxpar Direct Immune Profiling Assay and acquired with the Maxpar Direct Immune Profiling templates. |
Acquisition System |
|
Refresh Interval (sec) |
The frequency at which the plots update. NOTE Use the default setting (1 sec) for tuning and data acquisition. Adjust this setting on the recommendation of Fluidigm Service or Support for troubleshooting purposes. |
Save Files |
|
Events (Randomize, Normalize) MBPR SPR BgPR LMD IPPR TOF |
Select the file types you want to acquire. The default file selection includes the following files: • Events, randomized and normalized (FCS file for downstream analysis) • MBPR (mean beads per reading) • SPR (signal per reading) • BgPR (background per reading) • LMD (linear mode data, a compressed raw data file) For CyTOF Users: An LMD file type must be selected in Settings Templates used for data acquisition. A notification is displayed if the Template does not include an LMD file. Be sure LMD is a selected file type in the Template you want to use. For CyTOF Administrators: A warning is displayed when acquisition is started if either an LMD or events FCS file type is not included in a Template used for data acquisition. NOTE IPPR and TOF files are for troubleshooting purposes and do not need to be acquired. For more information about data files, see Choosing File Types. |
Event Detection |
|
Min Event Duration (pushes) |
The minimum number of consecutive pushes with signal greater than the Static Threshold required to trigger an event. The default setting of 10 pushes is recommended for data acquisition. Reduce this setting only if event length is very short (<10 pushes).
|
Max Event Duration (pushes) |
The maximum number of consecutive pushes with signal greater than the Static Threshold to trigger event detection. For example, if Max Event Duration is 150 pushes and 151 consecutive pushes are above the Static Threshold, an event is detected but not recorded in the FCS file. The default value is recommended for use.
|
Static Threshold (Dual Counts) |
The minimum total ion count per push required for event detection. All
pushes below the static threshold are excluded from event detection. The
default setting is 12. (For Helios™ users, this is equivalent to the lower
convolution threshold value of 400 in earlier versions of CyTOF Software
for Helios.)
During acquisition, view the Rain Plot (Data Display > Rain Plot). If signal from multiple channels contributes to noise that is higher than the threshold line on the Transient graph, increase the threshold.
|
Subtract Background |
Background is the average signal from individual channels between events. When selected, the background is subtracted per channel from event-triggering pushes. Using this setting is recommended, and it is selected by default.
|
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