Elispot for Rookies (and Experts Too)
Description
This book provides basic, simple, and logical explanations for choices to be made to run the best Elispot possible. It allows the newcomer to truly understand the best options for specific protocol steps, reagents and materials, and provides even the experienced Elispot user with insight into best practices. The techniques presented here are supported by the author's twenty-plus years of first-hand experience working with this assay, as well as the shared experiences of numerous colleagues and collaborators.
The enzyme-linked immunospot (Elispot) assay is a widely used technique to monitor cells on the single cell level for the release of analytes like cytokines, chemokines or immunoglobulins (antibodies), in response to particular stimuli. The most important feature of Elispot is its outstanding sensitivity, allowing the detection of specific cells in very low frequencies. The advantages of Elispot have resulted in it being widely adapted for use in research and translational applications in numerous fields including cancer, infectious diseases, autoimmunity, and transplantation.
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Content
Preface.- Overview.- Historic Overview.- Principles of Elispot.- Modifications.- Simplifications.- Coating Step.- Detection Step.- Adaptations.- Detection of specific antibodies.- Detection of multiple analytes in one well.- Dual color Elispot.- Fluorospot.- Summary.- Important Assay Details.- Reagents and Materials.- Elispot plates.- Antibody choices.- Enzymatic conjugates and substrates.- Fluorescent dyes.- Culture media.- Blocking buffers.- Washing buffers.- Important laboratory supplies.- Pipettes.- Filters.- Other supplies.- Automated Elispot reader.- Sample preparation.- Overview.- Whole blood and PBMC.- Blood draw and anticoagulants.- PBMC isolation.- Time frame between blood draw and PBMC isolation.- Immune cells obtained from tissue.- Freezing of cells.- Thawing of cells.- Cell counting and viability.- Overnight resting.- Isolation of sub-populations of cells.- Expansion of cells.- Planning the experiment.- Controls.- Negative controls.- Positive controls.- External controls.- Plate layout.- Timing.- The Elispot assay.- Coating.- Blocking.- Effector cell preparation.- Stimulant preparation.- Peptides and peptide pools.- Proteins and lysate.- Antigen presenters.- Control stimulants.- Reference samples.- Plating out cells and stimulants.- Incubation.- Removal of cells.- Spot development.- Enumeration of spots.- Data analysis.- Quality control.- Reporting guidelines.- Troubleshooting.- References.