The Immunological Toolbox Flashcards
To understand immune response we:
- Evaluate and quantify immune cell types in vitro and ex vivo.
- Characterize cell phenotypes
- Quantify effector molecules expresses on and by various cell types.
- Test the functional capacity of cells and molecules
- Determine cell interactions and fucntions in vitro and in vivo.
What’s the purpose of flow cytometry?
- To analyze fluorescently-labelled cells that emit light when they flow past lasers.
- It helps in distinguishing cell populations, evaluating cytokine production, testing cell viability, and determining trasnfection efficiency.
Flow Cytometry Spectrum Analysis
Focuses on the importance of spectreal overlap in designing flow cytometry panels to ensure accurate measurement of points of interest.
Visualization strategies and easly flow cytometry capabilities
Flow Cytometry Visualization Strategies
Effector molecules expression is detected inside the cells
Intracellular staining and cytokine secretion
Flow cytometry challenges and differente approches
- Early Flow Cytometry machines allowed measurement of up to five markes.
- Current technologies allow simultaneous measurement of up to 40 markers (requires proper compensation to prevent bleed over of fluorescent signals between channels).
- Mass cytometry approaches (using heavy metal labeled antiboides) measure up to 500 parameters can be complicated by metal oxidation whic reduces signal sensitivity.
Mass cytometry (CYTOF)
With CyTOF, overlap is minimized further, allowing for analysis of ~100 parameters.
Cell Sorting in Flow Cytometry
Flow cytometry can be used for sorting cells based on their expression of specific markers like CD8 and CD4, using charged-based separation.
* D1 (CD8) detected: Adds negative charge to cell. Cell is pulled into left tube.
* D4 (CD4) detected: Adds positive charge to cell. Cell is pulled into right tube.
Enzyme-Linked Immunosorbent Assays (ELISA)
Protein expression analysis
Direct ELISA: Measures binding of an antibody to a single analyte.
Indirect ELISA: Adding a second antibody to amplify the signal.
Sandwich ELISA: Determining the quantity of one analyte in a heterogeneous mixture.
Competitive ELISA: Measure of antibody affinity if you have a known thing that binds.
Western Blot
Steps of WB
ELISA vs. WB
Learn table
Flow Cytometry vs. ELISA vs. WB
Learn table
Immunoprecipitation
Isolation of proteins from a mix
- Immunoprecipitation: Allows to study protein-protein interactions, post-translational modifications, or other protein-related phenomena.
- Cell Lysis -> Antibody Binding -> Capture with Beads -> Washing -> Elution -> Protein Analysis.
Affinity Chromatography
Isolation of proteins from a mix
- It leverages the unique affinity of a target molecule for a particular substance (ligand) that is immobilized on a chromatography column.
- A highly specific method of purifying proteins, enzymes, antibodies, or other molecules from complex mixtures based on their specific binding interactions with a ligand.
You need to prepare the columns (beads)
Technique for measuring phagocytic capacity of macrophage or neutrophils
OVA
Quantitative PCR with reverse transcriptase (qRT-PCR).
Transcript (mRNA) analysis
Step 1: Reverse Transcription (mRNA -> cDNA)
Step 2: Quantitative PCR
RNA - sequencing (RNA-seq)
Transcript (mRNA) analysis
To perform whole-transcriptome analysis of samples.
CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats)
Manipulation of gene expression
- Takes advantage of a bacterial genome editing systems
- Faster, cheaper, more accurate, and more efficient than other genome editing methods.
Cre/lox System
Manipulation of gene expression
- Generate tissue-specific and inducible knockouts (control location and timing of gene expression).
- Turn transgene on or off.
- Track individual cells.
- Generate inversions or translocations.
- Report gene expression
- Study genes important to development.
Temporal control of knockouts
Manipulation of gene expression
Cre can be incorporated into a fusion protein with estrogen receptor 2 (Cre-ERT2)
* Cre-ERT2 is sequestered in the cytosol.
* Tamoxifen treatment allows Cr-ERT2 to enter the nucleus to excise floxed target sequence.
* Thus, reporter is only activated when tamoxifen treatment occurs.
