Flow cytometry Flashcards
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
What is compensation necessary for?
Give an example.
It controls for overlapping emission spectra of fluorophores
Ex: FITC overlaps in PE’s channel spectrum → cells that would have FICT-Ab only and no PE, would still appear a bit positive in the PE channel before compensation (false PE positive)
Opposit is not true → PE does not overlap in FITC’s channel
What different markers can we stain for with flow cytometry?
- Extracellular markers
- Detection of PTM
- Analysis of cell cycle phases, cell viability, cell proliferation
- Intracellular proteins (but have to kill, fix and permeabilize the cells for that)
What are other technics than flow cytometry can be used for cell separation (cheeper)?
Mostly used for blood cell separation
Density gradient centrifugation:
1. Plasma and platelets have the lowest density
2. Density of lymphocytes and monocytes < Ficoll → over Ficoll (PBMC or Buffy coat = peripheral blood mononuclear cells, lymphocytes + monocytes)
3. Layer on only Ficoll → neutrol, highly branched, hydrophilic polymer of sucrose which dissolves readily in aqueous solution
4. Granulocytes
5. Red blood cells (denser than Ficoll)
How does fluorescent activated cell sorting function?
- Normal flow cytometer
- Tract progress of each cell
- Break up the stream of sheath fluid into droplets (1 droplet = 1 cell) → droplets are given en electronic charge dependent on the fluorescence of the cell inside
- Deflection plates attract of repel the droplets/cells into max 4 different collecting tubes
Deflection plates = 1 positive on one side, 1 negative on the other side
What can be done if we want to sort cells for an intracellular marker rather than a cell-surface marker?
Fluorescence activated cell sorting using fluorescence reporters
To stain for intracellular protein requires cell fixation and permeabilization → no good for isolation of live cells
Possible to express a fluorescence reporter protein (GFP) under control of promotor or our gene or interest → live cells expressing GFP are then sorted
What is the difference between conventional and spectral flow cytometry?
Conventional: Predetermined channels are set to specific emission wavelengths for specific fluorophores
- Limited by number of available detectors and potential spectral overlap between fluorophores
Spectral: fluorescence signals are detected across the whole spectrum of wavelengths without predefined optical filters and detectors → simultaneous measurement of all emitted wavelengths → detailed spectral information for each individual cells → unmixing
In spectral, compensation ~ is called unmixing
Which are the polymorphonuclear and mononuclear leukocytes?
PMN leukocytes → Granulocytes (Neutrophils)
PBMC = peripheral blood mononuclear cells → monocytes (cytosolic granules, horseshoe-shaped nucleus) and lymphocytes (no granules, large round nucleus)
What is magnetic enrichment or depletion?
Magnetic enrichment → positive selection :
1. Ab is conjugated to magnetic beads
2. Ab binds to target on cells
3. Cells pass through a magnetic column → only non bound cells go through
4. Take magnetic field down → elution of the labeled cell fraction (damages a bit the cells, not great if want to reuse)
*Cheaper, more purity, simpler
Magnetic depletion → negative selection :
1. Non-target cells are magnetically labeled biotynilated + anti-biotin microbead
2. Cells passed through the column → only target cells pass through
*Good to have 100% intact cells, less purity, many Ab necessary
Flow cytometry is more expensive and complicated, but better to have 100% purity
Magnetic enrichement doesn’t give levels of expression → positive/negative with binding or not of Ab at a specific threshold of expression
What are 2 critical roles of dendritic cells?
- Tissue surveillance for infection
- Antigen presentation and T cell activation (bring antigens to the lymph nodes) → MHC peptide on DC interacts with TCR on T cells
What are the main functions/interaction of the MHC-peptide complex of dendritic cells?
What is the difference between MHC-I and MHC-II?
What makes dendritic cells special?
MHC-I:
- Found on surface of almost all nucleated cells
- Present proteins synthesized within the cells (viral/tumor antigens)
*DC can present exogenous antigens through MHC-I to CD8 cytotoxic T cells
MHC-II:
- Expressed on the surface of Antigen Presenting Cells (DC, Macrophages, B cells)
- Present peptides derived from exogenous antigens, engufed and digested by APC
*DC present extracellular antigens to CD4 T-helper cells through MHC-II
What is essential on dendritic cells (in addition to antigen) for T cell activation?
Co-stimulatory molecules (on the surface of DC) act as a 2nd signal:
- CD80 (B7.1)
- CD86 (B7.2)
- CD40
- OX40L
etc.
Checkpoint markers also modulate the signal (on DC):
- PDL1
- PDL2
etc.
How can microbes induce activation of dendritic cells?
Stimulation of Pattern Recognition Receptor (PRR) on DENDRITIC CELLS by microbial compounds induces cell activation → expression of co-stimulatory molecules
Best characterized family of PRR → Toll-like receptors (TLR)
Can have cell-surface TLRs (ligands are extracellular) and endosomal TLRs (ligands are dsRNA, ssRNA, CpG, etc.)
*link between innate and adaptative immunity → DC recognize an antigen and mediate the apropriate immune response
What is important to remember when using Bone Marrow Dendritic Cells (BMDC)?
It is a very simplistic model
What is the minimal air velocity in a Class II Biosafety Cabinet?
0.5 m/s
What is the effect of LPS-stimulation?
Might inhibit cell proliferation
Stimulates HSC by major inflammatory cytokines through TLR4 patway → generation of cytokines → stimulates differentiation and proliferation of cells?
Fluorochrome is PerCP-Cy5-5
-%Fluorochrome is PE
Spectral overlap is = 31.11
What does it mean?
PerCP-Cy5-5 Overlaps onto PE’s spectrum
The overlapping fluorochrome is in the Fluorochrome section, then one that is being overlapped on is -%Fluorochrome
What are the steps for dendritic cells tissue surveillance for infection?
- Antigen capture by DC
- Inflammatory cytokines secretion
- Loss of DC adhesiveness
- Migration of DC through the afferent lymphatic vessels
- Maturation of migrating DC
- Mature DC presents antigen to naive T cell (CD4+CD8+)
What are 2 methods for single-cell suspension?
- Mechnanical disaggregation
- Enzymatic digestion
*Depends on tissue, some cells might be lost in that process
What are different possible data presentation tools?
- Histograms (# cells/parameter x)
- Dot plots (2 parameters)
- Density plots (changes color with density)
What can be the importance of combining flow cytometry with imaging?
*See which markers are expressed and if there is a correlation with localization in the cell of an organelle
- Co-localization
- cell-cell interaction
- cell cycle and mitosis
- DNA damage and repai
- cell death and autophagy
What are to machines for magnetic enrichment or depletion?
- Manual magnet
- Automated cell separation unit
What are the main differences between FACs vs Magnetic Enrichment?
Complexity → FACS > MACS
Purity → FACS (>98%) > MACS (90-98%)
Specificity → high for both
Positive/negative selection → possible for both (low purity for MAC negative selection)
Multi marker selection → very limited for MACS
Sorting for distinct expression levels → only FACS
Sorting of cells with intracellular fluorescence → only FACS
Simultaneous sorting of different populations → possible for FACS, very limited and not simultaneous for MACS
What does CTRL stand for?
control
Similar as something identified as buffer only
How can you select for living cells in flow cytometry?
By staining and gating for DAPI
