The Immunological Toolbox

Question 1

To understand immune response we:

Answer 1

• 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.

Question 2

What’s the purpose of flow cytometry?

Answer 2

• 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.

Question 3

Flow Cytometry Spectrum Analysis

Answer 3

Focuses on the importance of spectreal overlap in designing flow cytometry panels to ensure accurate measurement of points of interest.

Question 4

Visualization strategies and easly flow cytometry capabilities

Answer 4

Question 5

Flow Cytometry Visualization Strategies

Answer 5

Question 6

Effector molecules expression is detected inside the cells

Intracellular staining and cytokine secretion

Answer 6

Question 7

Flow cytometry challenges and differente approches

Answer 7

• 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.

Question 8

Mass cytometry (CYTOF)

Answer 8

With CyTOF, overlap is minimized further, allowing for analysis of ~100 parameters.

Question 9

Cell Sorting in Flow Cytometry

Answer 9

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.

Question 10

Enzyme-Linked Immunosorbent Assays (ELISA)

Protein expression analysis

Answer 10

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.

Question 11

Western Blot

Steps of WB

Answer 11

Question 12

ELISA vs. WB

Learn table

Answer 12

Question 13

Flow Cytometry vs. ELISA vs. WB

Learn table

Answer 13

Question 14

Immunoprecipitation

Isolation of proteins from a mix

Answer 14

• 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.

Question 15

Affinity Chromatography

Isolation of proteins from a mix

Answer 15

• 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)

Question 16

Technique for measuring phagocytic capacity of macrophage or neutrophils

OVA

Answer 16

Question 17

Quantitative PCR with reverse transcriptase (qRT-PCR).

Transcript (mRNA) analysis

Answer 17

Step 1: Reverse Transcription (mRNA -> cDNA)
Step 2: Quantitative PCR

Question 18

RNA - sequencing (RNA-seq)

Transcript (mRNA) analysis

Answer 18

To perform whole-transcriptome analysis of samples.

Question 19

CRISPR
(Clustered Regularly Interspaced Short Palindromic Repeats)

Manipulation of gene expression

Answer 19

• Takes advantage of a bacterial genome editing systems
• Faster, cheaper, more accurate, and more efficient than other genome editing methods.

Question 20

Cre/lox System

Manipulation of gene expression

Answer 20

• 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.

Question 21

Reporter mice

Manipulation of gene expression

Answer 21

Benefit:
* Uncouples target gene expression from downstream analysis/effects.

Question 22

Temporal control of knockouts

Manipulation of gene expression

Answer 22

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.