The access to the services should be requested by sending a request to access to fcsc@uabmc.edu
Please include the following information.
Full Name (First, Last):
Blazer ID:
Email:
PI full name.
The access is provided within 24 hours.
Once access to FBS has been provided please fill the COMET Sample Submission Form.

• All runs of the COMET™ system are required to use the FSCS COMET Antibody Bank. This invaluable resource accelerates our progress by allowing our community of users to build upon each other’s success and expertise.
• Currently, the bank represents panels reactive to human, mouse, rat, and canine tissue sections with markers spanning the tumor microenvironment, neurology, and immunology.
• This bank is a continually expanding resource and is guided by the experimental needs of our users.
• See the most up-to-date database here. Please contact fcsc@uab.edu should you have questions.

• All COMET runs are Assisted.
• Optimization of markers takes on two forms: Mono/single antibody and Multiplex.

1. Mono-optimizations.
   • This is for new antibodies being added to the bank or testing of antibodies already existent in the bank but not yet tested on the desired tissue type or organism.
   • The detailed optimizations are broken down into 3 “characterizations” as guided by Lunaphore. The FCSC core has procured normal C57Bl/6 and human tissue arrays for this initial characterization. The requesting investigator must provide tissue types not reflected in this array.
   • Characterization 1: Does it work?
     Best guess concentration (usually on the high side) on tissues where it should express.
   • Characterization 2: What’s the best concentration/exposure?
     Approximately 3 concentrations to pick the best exposures and concentrations.
   • Characterization 3: How stable is the epitope?
     Initial stain, then serial elutions (5-20x) followed by identical staining.
2. Multiplex optimizations.
   This is for the optimization of a full multiplex panel of antibodies in the bank and has proven to work on the tissue of interest on your specific tissue of interest. Every tissue stains differently; some markers may need to be tweaked on new samples. This is a check before a full cohort is run. When successful, the exact staining conditions will be used on the multiplex panel experiment. All data is saved and can be used as one of the samples.

Users will be charged on a per slide basis depending on the number of (previously validated) antibodies that are used:

Users will also be charged for use of the instrument: $470/run. Each run takes about 24 hours and the instrument will process up to 4 slides at a time. All 4 slides must use the same combination of antibodies.

For example, if a user wants to image 7 slides using 8 antibodies, the price will be (7 x $560/slide) + (2 x $470/run).

Please consult with Harish Pal prior to scheduling the experiment so that a quote can be developed.

No. fo antibodies/slides                                 1 slide   2 slides     3 slides    4 slides
1-5 antibodies = 430/slide + $470 fee              $900      $665        $587        $548
6-10 antibodies = 560/slide + $470 fee          $1,030      $795        $717        $677
11-15 antibodies = 690/slide + $470 fee        $1,160      $925        $847        $807
16-20 antibodies = 820/slide + $470 fee        $1,290    $1,055       $977          $93

For additional, antibodies in the panel add $26/antibody/slide/concentration run in the total cost.

So for 30 antibodies panel with 20 cycles it should be ($26x30= $780 for antibodies/slide) + ($300 consumables/slide) + $470 Service fee/slide = $780 + $300 + $470 =$1550/slide.

New antibody validation

New antibodies must be validated and titrated prior to inclusion in an experiment. The Core will purchase previously untested antibodies according to user recommendations and will validate/titrate those antibodies on positive control slides provided by the user.

For example, a new antibody could be used at 3 concentrations on a positive control slide along with a panel of previously validated antibodies. Users would be charged for the run and for the number of antibodies (new antibody used at 3 dilutions counts as 3 antibodies) according to the pricing schedule above.

In another example, a user might want to validate multiple antibodies on a positive control slide.

Each antibody would be tested at 3 concentrations. Thus, if 5 new antibodies were tested at 3 concentrations each, the charge for the test panel would be based on 15 antibodies and a charge for the run according to the schedule above.

Please consult with Harish Pal prior to scheduling the validation so that a plan can be developed and a quote can be provided.

Sample can be anything that can attach to a standard microscopic slide and fit within the staining and imaging window of the microfluidic chip. Some examples are tissue sections, FFPE, snap frozen tissue, etc.

Where you place your sample matters. A model drawing of a slide is provided in figure 1. Anything within the blue box is an acceptable location for your sample. Thus, stars can be seen within the blue box as stars represent acceptable locations for your sample. In contrast, the triangles are located outside the blue box because they represent unactable locations for your sample.

The red square, in figure 1, represents a metal wire contained within the chip. Anything within this metal wire is safe.

The imaging window has the size of 12x12 mm. This imaging window will be directed toward the center of the slide. Thus, ensure that whatever you want to imaged is located at the center of your slide and can be contained within the 12x12 mm imaging window. Whatever part of your sample that is outside the 12x12 mm imaging window WILL NOT BE IMAGED. In figure 1, the 12x12 imaging window is represented by the yellow square.

Multiplex Immunofluorescence (mIF)
Three research institutes at UAB (O’Neal Comprehensive and Cancer Center, Immunology Institute, and I4ward fund) have demonstrated continuing support in the form of physical infrastructure, personnel expertise (dedicated staff in a shared resource facility), and financial investment into the support of the proposed multiplex immunofluorescent (mIF) imaging studies. This combined investment has resulted in the recent purchase of the COMET multiplex imaging system, the establishment of an optimized antibody bank, and computational support for the analysis of mIF images in collaboration with the Research Computing and Cheaha Cluster teams. These endeavors are run at or below cost (i.e., supported by other grant mechanisms) to maximize the impact, accessibility, and successful use of these technological investments. Detailed training and workflows have been generated and are made available on-demand to all UAB and collaborating researchers. These investments and the collaborative interactions between the Biospecimen Bank (clinical samples) and Animal Core (pre-clinical samples) demonstrate an ideal environment for completing the proposed aims.

EQUIPMENT
Lunaphore COMET Multiplexing System
Located within the Flow Cytometry and Single Cell Shared Resource on the UAB campus in the Shelby Research building. The COMET system is a self-contained liquid handler and microscopic image scanner in one fully modifiable program to optimize and validate three fluorophores each cycle. An optimized antibody bank and a BioGenex EZ-Retriever antigen retriever system support the COMET system. The fully trained core staff supervises all reagents, antibodies, protocol optimizations, and equipment operations.

Visiopharm Image Analysis Software
UAB investigators and core facilities have access to several seats of the full research license of Visiopharm analysis software. Access to 75TB of storage for each research faculty within the Research Computing core is provided to store multiplex scanned images for analysis. These systems come with several hours of detailed user support and training each month and access to on-demand, online overview training for all future and current users, supported by Dr. Carstens.