HSCI 442

EDTA

* used in blood storage
* anticoagulant
* chelates to ions in blood to prevent clotting

RPMI-1640

* Roswell Park Memorial Institute Medium
* is a cell culture media
* supports cell growth and vitality

FBS

* fetal bovine serum
* contains proteins and nutrients that support cell vitality

HBSS

* Hank's Buffered Salt Solution
* composed of inorganic salts and supplemented with glucose
* used to wash cells
* promotes cell vitality and maintains optimal pH (7-7.4)

PBS

* Phosphate Buffered Saline
* a physiological balanced salt solution used as a rinse in cell culture
* used in dilutions, washing cell suspensions, and rinsing
* maintains pH

D-PBS

* Dulbecco's Phosphate-Buffered Saline
* used for dilutions, rinsing cells and as a buffer
* maintains pH
* typically contains lower in phosphate concentration and may include calcium, magnesium, or chloride

cold D-PBS

- slows down the metabolic processes of cells and keep the cells from dying

RBC lysis buffer

* lyses erythrocytes in single cell suspensions of hematopoietic tissues (e.g., spleen and human peripheral blood) w/ minimal effects on lymphocytes
* contains ammonium chloride

trypan blue

* a viability stain used to differentiate dead cells (blue) from living cells (clear)
* binds to dead cells
* cell counter accounts for 1:2 dilution of cells in trypan blue

DMSO

* dimethyl sulfoxide
* cryoprotectant for cells
* penetrates the cell membrane by forming pores and thus prevents intracellular ice formation by reducing the water content inside the cell

blood smear steps

1. place a small drop of blood on a clean slide--hold a second slide at a 30-40 degree angle on the slide in front of the drop
2. maintain contact and move second slide back to contact the drop, let the blood spread across the second slide by capillary action
3. maintain firm contact with the bottom slide and push the top slide in one motion to produce the smear

Wright's stain

* a combination of eosin (red) and methylene blue
* allows differentiation between white and red blood cells

Flow Staining Buffer

- contains HBSS and Pen/Step solution (antibiotic)

live/dead cell stain

* dye binds to dead cells--allow them to be removed from the analysis
* light sensitive

Fc receptor blocking

* TruStain FcX PLUS anti-mouse CD16/32
* decreases non-specific antibody binding
* usually added when staining lymphocytes for flow cytometry

anti-CD45R/B220 antibody

binds to B cells

anti-CD8 antibody

binds to CD8 T cells

antibody titration

allows researchers to determine the amount of necessary antibody needed for optimal stain resolution

paraformaldehyde

* PFA- formaldehyde in aqueous solution
* fixes cells in place

anti-CD4 antibody

binds to CD 4 T cells

anti-NK antibody

binds to NK cells

cyanine dye blocking solution

* True-Stain Monocyte Blocker
* blocks non-specific binding of PE and APC

compensation controls

* corrects for fluorescence spillover
* removes the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye

FMO

* fluorescence minus one
* way of creating positive gates
* samples are stained with all the fluorophores in your panel, minus one of them

Luria-Bertani

* lysogeny broth
* rich media used for bacteria growth

Escherichia coli O127:H6 strain E2348/69

spectrophotometer

- measures bacteria concentration at optical density 600 nm- OD(600 nm) = 1x10^8 cells/ml

DMEM

* Dulbecco's Modified Eagle's Medium
* supports cell growth
* lower glucose concentration than RPMI

ovalbumin

egg white protein

sodium azide

* preservative
* prevents microbial contamination
* can be added to produce good storage buffer

IgG

most abundant antibody found in the plasma

IgG1

most abundant and versatile

IgG2

* 1:1 mix of IgG2b and IgG2c
* no IgGa because mice lack the gene encoding antibody
* responds to bacterial capsular polysaccharide antigens

Tween (PBS-T)

* helps to prevent non-specific antibody binding
* to remove unbound antibody

Casein (PBS-C)

* blocking buffer
* used for blocking excess binding sites on membrane and microplates in antibody-based detection

alkaline-phosphatase (AP)

* enzyme bound to antibody in ELISA (secondary antibody)
* uses the substrate p-nitrophenyl phosphate which produces a water-soluble yellow reaction product

p-nitrophenyl phosphate

* pNPP- substrate for AP
* produces a water-soluble yellow reaction product
* light sensitive reaction

ELISA

* enzyme-linked immunosorbent assay
* can be indirect or direct

immune system: anatomic barriers

* skin
* oral mucosa
* respiratory epithelium
* intestine

innate immune defenses

* complement
* macrophages
* granulocytes
* natural killer cells

adaptive immune defenses

* b cells
* t cells

leukocytes

white blood cells

thrombocytes

platelets

erythrocytes

red blood cells

blood smear

a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examine microscopically

purpose of blood smears

* look for parasites (e.g., malaria)
* hematological problems (e.g., sickle cell)
* cancer (e.g., leukemia)
* effects of chemotherapy

neutrophil

**role:** phagocytosis and killing of microorganisms

* granulocyte
* multi-lobed nucleus (normally 3-5)
* numerous small/purple granules
* constitutes 50-70% of white blood cells (i.e., most abundant)
* stay in circulation and are not present in healthy tissue
* generate inflammatory response

eosinophil

**role:** killing of antibody-coated parasites through release of granule contents

* bi-lobed nucleus (normally red/pink)
* numerous closely packed orange/red granules
* 1-4% of white blood cells
* increased in parasitic infections

basophil

**role:** controlling immune response to parasites

* densely packed, large purpose cytoplasmic granules
* kidney-shaped nucleus (difficult to see)
* constitutes less than 1% of white blood cells
* increased during asthma

monocyte

**role:** circulating precursor cell to macrophage

* very fine, dark cytoplasmic granules
* kidney-shaped nucleus
* 2-10% of white blood cells
* round-ish shape
* located in blood
* continually turning over to macrophages in adulthood

macrophage

**role:** phagocytosis and killing of microorganisms; activation of t cells and initiation of immune response

* large, irregular shape
* very large cytoplasm
* small nucleus
* cytoplasmic granules present
* found in tissue; become resident in tissues during childhood

large lymphocyte

**role:** kills cells infected with certain viruses

* cytoplasmic granules
* irregular shaped nucleus
* natural killer cell

small lymphocyte

**role:** production of antibodies (b cells); cytotoxic and helper functions (t cells)

* little visible cytoplasm
* large, round nucleus
* small
* uniformly shaped
* no granules

thick blood smear

* too large blood drop
* angle too high

thin blood smear

* too small blood drop
* angle too low

dependents of good blood smear

* size of the blood drop
* angle applied to the spreader
* speed/steadiness in pushing the spreader

qualities of good blood smear

* takes up 1/2-3/4 of the entire slide
* tongue-shaped
* lateral edges of smear are visible
* smooth without (many) irregularities

bi-variate dot plots

* can see relationship between markers
* can see sub-populations in 2 dimensions instead of one

forward and side scatter

* show in linear scale; **exceptions**: small things like bacteria, extracellular vesicles, and nuclei
* provides gating of lymphocytes/exclusion of dead cells

flow cytometry: log scale

* use for dynamic ranges
* compresses scale to get nice, round populations

live/dead gating

* removes dead cells from sample
* dead cells can soak up antibody and give false positives

double positive events

can occur due to debris in sample or presence of dead cells

fluorescence parameters

* uses log scale; exception for low single increase
* provides gating of specific cell markers

median fluorescence intensity (MFI)

* used to assess levels of target protein expression
* use median for log data
* use mean for linear data only

MFI fold-change

* used to compare the expression level of antigen/marker between samples
* fold change = MFI (sample) / MFI (control) = times increase in marker
* can compare fold-change in MFI between treatments/samples

flow cytometry antibody titration

* ensures cells are not over or under-stained
* over-stain = non-specific staining
* under-stain = insufficient detection of target/molecule

flow cytometry

* measurements of cells/particles in liquid suspension
* simultaneous measurement of one or more characteristics of single cells as they pass through a laser

forward scatter

a measure of the relative cell size

side scatter

measures the relative complexity of the cell (e.g., granules, nucleus, etc.)

flow cytometry antibodies

* artificially conjugated to fluorochromes
* bind to specific surface molecules on target cells

longpass (LP) filter

* allow wavelengths longer than the filter rating to pass through
* reflects shorter wavelengths

bandpass (BP) filter

* allow a relatively narrow range or band of light to pass through filter
* wavelengths outside range are reflected
* typically designated by 2 numbers

flow cytometry steps

1. emission of conjugated ab
2. detection of fluorescence
3. conversion to light into voltage
4. measure height, area, and width of light pulse
5. FCS file with raw data generated
6. data plotted

flow cytometry histograms

* shows the distribution of values for a specific parameter
* cannot see the relationship between 2 populations
* can miss sub-populations that have similar values in one parameter
* often used for MFI (represents half way mark of one population)

FMO

* fluorescence minus one
* controls are samples stained with all the fluorophores, minus one of them
* used to set the upper boundary for background signal using control, and thus can identify and gate positive populations in multicolor experiments
* shows the background and contributions from neighbouring fluorescence spillover
* can create gates with control and then apply to test sample

fluorophore spectrum

hematopoietic stem cell

cell by which all lymphoid cells are derived

doublet discrimination

* helps remove events that are 2 or more cells stuck together
* reduces the contribution to false positive or double positive events
* set axis to FSC-A vs FSC-H, FSC-A vs FSC-W, SSC-A vs SSC-W, or SSC-A vs SSC-H

positive control

* standardize gating procedure and observe staining profile
* treated to induce positivity
* useful for rare positive populations or when expression is variable between sample

biological controls

* e.g., Stim vs Unstim, T0 vs Time Course, Treated vs. Untreated
* any control you need to prove your hypothesis?

unstained control

used to evaluate inherent background and autofluorescence

common gating control

* FMO control
* positive control
* biological control
* unstained control

when is compensation required

when there is spectral overlap between 2 or more fluorophores

requirements when there is spectral overlap between fluorophores

* compensation
* for all fluorophores and live/dead stain
* FMO controls

general gating strategy

1. lymphocytes
2. FSC single cells
3. SSC single cells
4. live cells
5. positive population

phagocytosis

* involved in innate response
* process in which phagocytes recognize, ingest, and kill pathogens
* pathogen uptake is usually through membrane receptors (e.g., complement, FC, scavenger receptor)

phagocyte

cell which ingests and kills invading pathogens

* macrophages (mononuclear phagocytes)
* neutrophils (polymorphonuclear cells; PMNs)
* dendritic cells

Fc receptor

* located on phagocytes
* activated by IgG1 and IgG3 antibodies bound to pathogen
* interaction enables or accelerates phagocytosis

3 cautions while working with spectrophotometer

1. avoid bubbles down in the cuvette


1. can affect the wavelength measurement
2. avoid touching the bottom of the cuvette to prevent smudges
3. throw out each use cuvette--they are not sterile

antibody hypervariable regions (complementary determining region)

* 3 heavy chain regions
* more variability than light chain
* HCDR3 has greater variability due to location in antigen binding region (middle/centre)
* 3 light chain regions
* less variable than heavy chain
* LCDR3 has greater variability due to location in antigen binding region (middle/centre)
* total of 6 binding interactions per antibody arm

antibody-antigen binding shapes

* hapin
* a
* antigen nestles into antibody
* for small molecules
* peptide
* b and c
* antibody forms a groove/canyon
* dimple/crevasse
* d
* antibody points out towards antigen

epitope

site on antigen to which the antibody binds

paratope

imprint on antibody of where the antigen was bound

epitope shapes

1. **conformational determinant**
2. **linear determinant**
3. **neoantigenic determinant**

conformational determinant

* binding is determined on antigen folding; folded peptide chain affects 3D spacing of residues that for epitopes
* non-linear; does not need to be a single protein (could be a quaternary structure)
* ab-ag binding is lost with denaturation

linear determinant

1. epitope is in same line sequence; does not depend on folding and does not need to be in consecutive sequence
2. accessible: ab can access epitope while antigen is folded
3. inaccessible: ab cannot access epitope while antigen is folded; only accessible through denaturation
4. ab-ag binding is not lost with denaturation

neoantigenic determinant

1. epitope does not exist in native protein/antigen
2. epitope is generated through a process that alters the structure, makeup or presentation of the protein (e.g., proteolysis)

adjuvants

added to vaccines to promote a greater immune response to vaccination

* Alum
* CpG-ODN

\*\* increase immunogenicity with slow release and when combined with bacteria

CpG oligonucleotides (ODNs)

* synthetic oligonucleotides
* contain unmethylated CpG dinucleotides in specific sequences (CpG motifs)
* activate toll-like receptor 9, leading to strong immunostimulatory effects

indrect ELISA required reagents

* antigen
* pure or semi-pure
* test solution containing antibody
* enzyme conjugate that binds Ig of immunized species