How are polyclonal antibodies produced?
What are the advatanges?
- Immunization of an animal with antigen
- Stimulates production of Ab that recognize the antigen
- Collect serum to obtain polyclonal Ab mixture
*Different immunoglobulins bind to the antigen at different sites and different affinities
*If buy another batch → might not get same binding/affinity as previous batch
Works for immunofluorescence, Western Blot, Co-IP because all have the same constant region
What are 2 methods for antibody production?
- Polyclonal Antibodies
- Monoclonal Antibodies
How are monoclonal antibodies produced?
What are the advantages?
- Isolate an Ab-producing cell (B cell) isolated
- Fuse it with tumor cell to generate immortalized hybridoma cells
- Screen hybridoma cell lines to identify the ones secreting Ab with desired specificities
*These lines can be maintained in culture to produce the unique monoclona Ab indefinitely
Used for ELISA, Flow cytometry (most of the time)
What are the 2 major types of flow cytometry machines?
- Analyzers → analyse cell composition
- Sorters → purify rare cell types
What does the flow cytometry machine measure?
- Forward and side scatter of the laser light
- Intensity of the emitted fluorescent light
FSC → cell size (lymphocytes are smaller)
SSC → granularity (granulocytes > monocytes >= lymphocytes)
*To measure mutlple parameter → bind different markers with different fluorophore labelled Ab
What is gating meant for?
Meant for displaying data for only a subset of cells found in a non-homogenous sample
What are isotype controls?
Why are they important?
Isotype control = Ab raised for a cell type NOT in the cell assessed
They control for non-specific Ab binding → should match test-Ab in:
- host species
- isotope (igG1 or igG3, etc.)
- fluorophore conjugation
- concentration
Important when analyzing:
- Immune cells with high Fc-receptor expression (bind to the constant region without specificity, found in DC, macrophages, B cells)
- Low expressed or poorly characterized markers (to validate results)
What different cell features/type of stainings can we do?
- Detection of intracellular proteins → cytokine production
- Detection of post-translational protein modifications → phosphorylations
- Analysis of cell cycle phase, cell viability, cell proliferation
What is required for intracellular protein phosphorylation staining (F.C.)?
To stain for intracellular protein cells need to be fixed + permeabilized → kills the cells
Chose an Ab that binds specifically for when a site is phosphorylated, not otherwise
What are the difference between western blots and flow cytometry?
Both are ways to analayze intracellular proteins in cells:
Western blots provide average data vs Flow provides single cell data
Western requires less specific Ab bc proteins are resolved by MW (flow = 1 reading/cell)
In Western, all sites are available because cell components are all separated, in flow, some binding sites might not be available (false negative)
How can flow cytometry be used to assess cell cycle?
- Propidium Iodide Staining for DNA content inside the cell → peak at G0/G1 (diploid organisms), smaller peak around G2/M (double diploid), S phase in the middle, less DNA content that G1 = apoptotice cells
*Higher peak at G2/M correlates with more proliferation, and inverse correlates with more quiescence - BrdU Incorporation analysis:
- BrdU (thymidine analog) is incorporated into DNA during S phase
- Can measure the # cells that went through S phase during exposure - Cell proliferation dye dilution assay:
Cells from original sample are stained with the dye → dye is diluted by 1/2 every time the cell divides
What is the volume of solution in 1 square of the hematocytometer?
10^-4 mL
What are CD11b and CD11c markers for?
CD11b (integrin alpha, member of integrin cells adhesion molecules) → marker for certain myeloid lineage cells → plays a role in immune signaling, phagocytosis, cell adhesion, migration
ex: monocytes, macrophages, dendritic cells, NK cells, granulocytes are all CD11b+
CD11c (integrin alpha, member of integrin cells adhesion molecules) → predominant expression on the surface of dendritic cells
What are CD40, CD80 and CD86 markers for?
*Activation and co-stimulatory markers
CD40 → TNF receptor superfamily on dendritic cells, interaction with ligand on T cells → positive signaling → expression of CD80/86 + production of IL-12 → activation, proliferation and differentiation of T cells
→ mediate innate and adaptative immune response
CD80/86 → co-stimulatory molecules on surface of APC, interact with CD28 or CTLA-4 on T or B cells
→ Provide second signal for further T cell activation (after TCR engagement with MHC)
→ Enhances T cell proliferation, cytokine secretion, enhance adaptative immune response
How does fluidics work?
Fluidics → system responsible for transporting the sample fluid containing the particles trough the instrument in a controlled manner
- Sustained cell suspension required (because clumps can clog the fluidics system)
- Taken up by machine and forced in a narrow stream of sheath fluid (~PBS)
- Passes in front of a system of lasers and photodetectors → analysis of 1 cell at the time
What are the 2 main machine components in flow cytometry ?
- Fluidics
- Optics → lases and photodetectors
- Software/data processing
How does the optics part of flow cytometry function?
Laser light of appropriate wavelength excites fluorophore on the cells and the intensity of the emmited light is measured
- Lasers = light source → emit at a specific wavelength (choice of laser depends on the fluorescent marker on the Ab marker)
- As the particle passes through the beam, interact with laser beam in different manners → FSC, SSC
- A photodetector in the forward direction detects the FSC → size
- A photodetector positioned at the side angle collects the side scattered light → granularity
As the particle is labeled with a fluorescent marker, it emits fluorescence when excited by a laser light
How are different fluorophore selected?
Fluorophore = fluorescent markers on Ab
All have to able to be excited by the chosen laser for 1 specific property → have different emission properties when excited by the same blue laser (different peak emission)
How can expression of many cell markers be measured simultaneously?
Multiparameter analysis:
Unse many fluorophore with different excitation and emission properties on the same sample
What does it mean to have 2 peaks in an emission histogram?
Have 2 different cell populations
What does it mean for a T cell population to be double positive or double negative for CD4 and CD8?
In the thymus development, immature T cells are first CD4-CD8-, then they are CD4+CD8+ (short lived/mature/no TCR signal). Following TCR signal, they differentiate into either CD4+ OR CD8+
In the thymus → mostly double positive
In Lymph nodes, blood, spleen → moslty CD4+ OR CD8+
In a cell sample from the lymph nodes, what would it mean to have a peak/dark patch in the double negative section (CD4-CD8-)?
The report for the population of non T cell in the lymph nodes
What is the importance of an isotype control?
When is it the most important?
Isotype control Ab staining → controls for non-specific Ab binding
It involves using Ab of the same isotype (ex: IgG vs IgA) as the Ab used, but specific for an antigen you shouldn’t have in your cell
Tells us if what we see is a true positive of just positive background → isotype control should be much lower than you positive result (closer to unstained control)
Mostly important when analyzing:
- Immune cells with high Fc-receptor expression (B cells and immune cells might bind the constant region of the Ab)
- Low expressed or poorly characterized markers
What should match between the isotype control antibody and the test-antibody?
- Same host species (rabbit, mouse, etc.)
- Same isotype (ex: IgG1 or IgG3 or IgG2a or IgM, etc.) → same constant region
- Fluorophore conjugation → same fluorophore is conjugated to the Ab
- Concentration
