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&rct=j&q=bd%20elispot%20instruction%20manual&source=web&cd=1&ved=0CC0QFjAA&url=http%3A%2F%2Fwww.bdbiosciences.com%2Fdocuments%2FElispot_set_instruction_manual.pdf&ei=4vsyT_PJN6HniALL7sTBCg&usg=AFQjCNE4atGDuzIzWmADJFUOqrCWyzX8PQ&cad=rja 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 > 2 × 106 cells/ml). . Add 100 µl/well of each cell suspension to ELISPOT plate microwells. >> 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. Best regards,  Kurt Whittemore
 * Coating Antibody
 * 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.
 * Store plates at 4ºC overnight.
 * Blocking
 * Discard Coating Antibody. . Wash wells 1× with 200 μl/well Blocking Solution*.
 * Add 200 μl/well Blocking Solution and incubate for 2 hr at room temperature.
 * Cell Activation
 * 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.
 * Prepare cell suspensions at different densities, (eg, 1 × 105cells/ml -
 * 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.
 * 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.
 * 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
 * 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.
 * 3X PBST 2 min at RT
 * 2 hr RT incubation with biotinylated antibody (100 uL). Add 40 uL biotinylated antibody to 10 mL 10% FBS in PBS
 * Prepare streptavidin mixture (100 uL streptavidin compound to 10 mL 10% FBS in PBS)
 * Wash 3X PBST 2 min
 * Add 100 uL of streptavidin mixture
 * Incubate 1 hr RT
 * After 30 min of the 1 hr incubation, bring 11 mL substrate buffer to RT in drawer in dark
 * Wash 3X PBST 2 min
 * Wash 2X PBS 2 min
 * After 1X wash with PBS add 11 drops chromagen into 11 mL substrate buffer
 * Add 100 uL substrate solution to plate
 * Make sure to use a clean reagent reservoir for this solution. Note about mistake 4-10-12
 * Develop 15 min RT
 * 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))
 * Stop the reaction by washing with H20.
 * Shen likes to wash his plate by shaking it in a bucket of H20 at this step.
 * 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
 * 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).
 * Place plate into opaque whit adapter by immunospot machine (maybe in a drawer)
 * Use the image acquisition for taking pictures, and the immunospot software for analysis (computer is slow so wait)
 * 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
 * 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
 * Turn off Immunospot machine
 * Get data off computer
 * 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.

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