BD ELISPOT Set

2013-12-01

azim58 - BD ELISPOT Set

BD ELISPOT Set

Version of Protocol also found here
S:\Research\Mol_Biol_Tech\Protocols\ELISPOT

Link to Instruction Manual:
http://www.google.com/url?sa=t&amp;rct=j&amp;q=bd%20elispot%20instruction%2
0manual&amp;source=web&amp;cd=1&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.
bdbiosciences.com%2Fdocuments%2FElispot_set_instruction_manual.pdf&amp;ei=4
vsyT_PJN6HniALL7sTBCg&amp;usg=AFQjCNE4atGDuzIzWmADJFUOqrCWyzX8PQ&amp;cad=rj
a
Instruction Manual
"C:\kurt\storage\CIM Research Folder\DR\2013\2-5-13\data_download\Annexin
V Apoptosis Kit APC Documentation.pdf"

Annotated ELISPOT Current Protocols in Immunology Document
"C:\kurt\storage\CIM Research
Folder\DR\2013\2-5-13\data_download\2127\Elispot_set_instruction_manual.pdf
"

Materials
BD ELISPOT SET which comes with many reagents and the plates
detection substrate which needs to be ordered separately
blocking buffer: 10% FBS in PBS
TBST

Protocol

* Coating Antibody
  o Dilute capture antibody in PBS pH 7.2 (see Certificate of
    Analysis provided with each BD™ ELISPOT Set for antibody dilution
    information). Add 100 µl of diluted antibody solution to each well
    of an ELISPOT plate.
  o Store plates at 4ºC overnight.

* Blocking
  o Discard Coating Antibody. . Wash wells 1× with 200 μl/well
    Blocking Solution*.
  o Add 200 μl/well Blocking Solution and incubate for 2 hr at room
    temperature.

* Cell Activation
  o Discard Blocking Solution. Prepare mitogen or antigen, diluted in
    complete medium (eg, RPMI 1640 with FBS, Pen/Strep, and L-glutamine).
    Add 100 µl/well to ELISPOT plate.
  o Prepare cell suspensions at different densities, (eg, 1 ×
    105cells/ml -
  2 × 106 cells/ml). . Add 100 µl/well of each cell suspension to
  ELISPOT plate microwells.

- Pulse target cells with peptide if necessary
  - Add T cells. Make sure to add ConA (1 or 2 ug/mL final conc.) to
    positive control T cell samples.
  o Replace lid. Incubate ELISPOT plate at 37ºC, in a 5% CO2 and
    humidified incubator. The duration of the incubation time will vary
    (eg, 2 hr – 24 hr (Shen often does 48 hours)). Specific activation
    conditions will vary, depending on cell type, kinetics, and analyte
    of interest. . Please see Certificate of Analysis provided with each
    BD™ ELISPOT Set for assay conditions, suggested cell types, and
    incubation times of suggested positive controls. After step 7,
    aseptic conditions are no longer needed.
    Note: Cells may be diluted in a regular tissue culture plate starting
    at 105/well in triplicate wells with 1:3 or 1:4 serial dilutions down
    the plate, then transferred to the ELISPOT plate.
    - May want to place a note on incubator door stating "do not
      open and close door" so that the plate is not disturbed and does
      not have streaks and ambiguous spots later. Also do not stack
      multiple ELISPOT plates in incubator.

* Detect cytokines
  o Wash plate 2X H20 200 uL 5 min
    - Shen likes to wash his plate by shaking it in a bucket of H20
      at this step.

o 3X PBST 2 min at RT
  o 2 hr RT incubation with biotinylated antibody (100 uL). Add 40 uL
    biotinylated antibody to 10 mL 10% FBS in PBS
  o Prepare streptavidin mixture (100 uL streptavidin compound to 10
    mL 10% FBS in PBS)
  o Wash 3X PBST 2 min
  o Add 100 uL of streptavidin mixture
  o Incubate 1 hr RT
  o After 30 min of the 1 hr incubation, bring 11 mL substrate buffer
    to RT in drawer in dark
  o Wash 3X PBST 2 min
  o Wash 2X PBS 2 min
  o After 1X wash with PBS add 11 drops chromagen into 11 mL
    substrate buffer
  o Add 100 uL substrate solution to plate
    - Make sure to use a clean reagent reservoir for this solution.
      Note about mistake 4-10-12

o Develop 15 min RT
  o Dilute strongly reacting wells such as the ConA control wells by
    repeatedly diluting with H20 (e.g. 50 uL can be added, 100 uL of H20
    removed, and this can be repeated several times (maybe about 3))
  o Stop the reaction by washing with H20.
    - Shen likes to wash his plate by shaking it in a bucket of H20
      at this step.

o Air-dry plate at room temperature for 2 hr or overnight until it
    is completely dry. Removal of plastic tray under plate will
    facilitate drying. Store plate in a sealed plastic bag in the dark,
    until it is analyzed.

* Image ELISPOT plates
  o Turn on immunospot (located in IDV) and then computer
    - Note that I think the Immunospot machine belongs to the lab
      of Dr. Jacobs. Karen Kibler is the lab manager of this lab, and can
      grant card access to the lab (Karen.Kibler@asu.edu).

o Place plate into opaque whit adapter by immunospot machine (maybe
    in a drawer)
  o Use the image acquisition for taking pictures, and the immunospot
    software for analysis (computer is slow so wait)
  o Open Image Acquisition
    - Easy Scan Wizard -> M200 white plate -> Save in D:\My
      Plates\Some_username.
      @ Eject-> load plate with A on side of sticker->Name plate
        (just click No if there is a message asking "Do you want to
        accept this new loas your default position)->Scanning options->No
        Auto center->1024X1024 resolution->Preselect the wells you want
        (Don't close the "Plate Navigator" window so that you can eject
        the plate later->Start scanning
      @ Note that the camera is at a slight angle so some of the
        well is not showing

- Eject after scanning by clicking the eject button in the
      "plate navigator window"->Cancel plate naming->Close software and
      plate holding tray will close automatically
    - take plate adapter off

o Open Immunospot
    - Automated count ->red
    - load plates ->username->plate name (make sure box has a
      checkmark in it)->resolution 1024X1003
    - Next
    - Step 2 of 5: Defining parameters -> fine tune parameters ->
      sensitivity -> you can audit spots to remove some -> start autocount
    - Close software

o Turn off Immunospot machine
  o Get data off computer
  o Note that spots are typically represented in the form of a
    histogram with spots per/(10^6 Splenocytes) on the y axis and the
    different samples on the x axis. Each sample would typically have
    error bars associated with it.

Best regards,
Kurt Whittemore

Graduate Student
Arizona State University
BIODESIGN INSTITUTE
Center for Innovations in Medicine
1001 S McALLISTER AVE
TEMPE, AZ 85287

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