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Macrophage > Techniques > Flashcards

Techniques Flashcards

1
Q

Fixatives used for routine microscopy…

A

Buffered isotonic solution of 4% formaldehyde or 2% glutaraldehyde

React with amine groups (NH2) of tissue proteins.

Glut: because it is a dialdehyde, which can cross-link proteins

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2
Q

Osmium Tetroxide (osmic acid)

A

2nd part of a double fixation procedure (glutaraldehyde being the 1st step) has become the standard procedure in preparations for ultrastructural studies.

Tetrahedral structure and nonpolarity allow it to penetrate charged cell membranes.

Stabilizes proteins w/o destroying structural features, binds to phospholipid head regions and prevents coagulation during alcohol dehydration

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3
Q

Basophilic

A

Tissues that stain more readily with basic dyes. Tissue components that ionize (create electrostatic salt linkages) and react with basic dyes do so because of acids in their composition.

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4
Q

Examples of basic dyes

A

Toluidine blue
Methylene blue
Hematoxylin

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5
Q

Basophilic tissue components

A

Nucleic acids, glycosaminoglycans, acid glycoproteins

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6
Q

Acidophilic

A

Dyes that stain acidophilic components such as mitochondria, secretory granules and collagen

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7
Q

Example of a acidophilic dye

A

Eosin

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8
Q

Resolving power

A

The smallest distance between two particles at which they can be seen as separate objects the quality of the image– it’s clarity and richness of detail– depends on the microscopes resolving power.

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9
Q

Resolving power of a light microscope

A

~ 0.2 um

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10
Q

Resolving power of electron microscope

A

0.1 nm although in practice 3nm is the best

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11
Q

Chemical fixation

A

Tissue samples are immersed in solutions of stabilizing or cross-linking agents called fixatives to avoid tissue digestion by enzymes present within the cells, or by bacteria to preserve the structure and molecular composition

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12
Q

Primary cell cultures

A

Cells that are cultured directly from a subject. Most have a limited lifespan. Cells may be released from soft tissues by enzymatic digestion such as collagenase, trypsin or pronase which break down ECM

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13
Q

Immortalized cell line

A

A cell line that has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification. This process is called transformation and may convert a normal cell into a cancer cell

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14
Q

Periodic acid-Schiff (PAS) reaction

A

Stain for polysaccharides like glycogen in the liver

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15
Q

Phalloiden

A

Stains actin when conjugated with fluorescent dyes

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16
Q

Polyclonal Antibodies

A

When several groups (clones) of B lymphocytes of the animal that was injected with protein X recognize different parts of protein X and each group produces an antibody against each part.

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17
Q

Monoclonal antibody

A

Individual lymphocyte clones (B lymphocytes fused with tumor cells i.e. Hybridomas) can be isolated in culture separately so that different antibodies against protein X can be collected separately.

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18
Q

Advantages to using a monoclonal antibody vs a polyclonal antibody

A

Monoclonal can be selected to be highly specific and to bind strongly to the protein to be detected. Therefore there is less specific binding.

Hybridomas provide immortal cell lines with ability to produce unlimited quantities of specific antibodies

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19
Q

Advantages to using a polyclonal antibody vs a monoclonal antibody

A

Can help increase signal produced by target protein as antibody can bind to more than one epitope

Less sensitive to antigen changes

Useful when nature of antigen is unknown

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20
Q

Direct method of immunocytochemistry

A

The antibody must be tagged with an appropriate label.

Ex: fluorescent compound, gold particles

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21
Q

Indirect method of immunocytochemistry

A

1) antibodies to protein of interest (human) must be produced in an animal of another species (eg. rat)
2) immunoglobulin from a noninjected rat must be injected into the animal of a 3rd species (eg. Goat) in order to induce antibody production to rat immunoglobulin
3) these goat antiantibodies (fluorescently conjugated) bind to the rat antibody that has previously recognized the human protein that can then be visualized.

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22
Q

Gel Electrophoresis/Western Blot

A

Isolation of Proteins
1) mixtures of proteins are obtained from homogenized cells and treated with a strong detergent (Sodium dodecly sulfate) and mercaptoethanol to unfold and separate the protein subunits

2) samples are put in wells of a polyacrylamide gel and submitted to an electric field, with proteins migrating along the gel according to size and shape.
3) a mixture of of proteins of known sizes is added to a well as a reference to identify molecular mass of other proteins

Detection & Identification of Proteins

1) all proteins stained the same color (bromophenol blue); color intensity is proportonal to protein concentration.
2) proteins can then be transferred from the gel to a nitrocellulose membrane. The membrane is incubated with a labeled antibody made against proteins that may be present in the sample (immnoblot)

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23
Q

Distortions & Artifacts caused b tissue processing

A

Shrinkage produced by fixative or ethanol

Artificial spaces due to loss of molecules (glycogen and lipids)

Precipitates of stain

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24
Q

Macrophage and neutrophil metabolism shift while in an anoxic, inflammatory environment

A

Oxidative to glycolysis

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25
Q

Integrins

A

Proteins of the plasma membrane that are linked to cytoplasmic cytoskeletal filaments and to extracellular molecules allowing for constant exchange of influence both ways

26
Q

Membranes are only visible using _________ because __________

A

Electron microscope

They range from 7.5 to 10 nm in thickness

27
Q

___________ breaks up the close packing of the phospholipid long chains, and this disruption makes the membrane more fluid

A

Cholesterol

28
Q

Proteins make up __% of the plasma membrane

A

50%

29
Q

Proteins of the plasma membrane include:

A

Integral Proteins: directly incorporated within the lipid bilayer

Peripheral Proteins: exhibit a looser association with membrane surfaces

30
Q

Receptors

A

Glycoproteins and glycolipids with carbohydrate moieties that project from the external surface of the plasma membrane and participate in important interactions such as cell adhesion, recognition, and response to protein hormones

31
Q

Fluid-Phase Pinocytosis

A

Small invaginations of the cell membrane form and entrap extracellular fluid and anything in solution fluid. Most fuse with lysosomes but some move to the surface opposite their origin.

“Cell-drinking”

32
Q

Receptor-Mediated Endocytosis

A

Binding of a ligand with high affinity to its specific receptor causes widely dispersed receptors to accumulate in coated pits.

33
Q

The major polypeptide of coated pits

A

Clatherin

34
Q

Endosomes are acidified by

A

ATP-driven H+ pumps

35
Q

Phagocytosis

A

Mechanism by which cells incorporate and remove foreign bacteria, Protozoa, fungi, damaged cells, and unneeded extracellular constituents via a phagosome

36
Q

Exocytosis

A

The term used to describe the fusion of a membrane-limited structure with the plasma membrane, resulting in the release of its contents into the extracellular space without compromising the integrity of the plasma membrane.

An increase in cytosolic Ca+ often triggers Exocytosis

37
Q

G proteins bind to…

A

… Guanine nucleotides

38
Q

Activation of G protein

A

First messenger binds to a receptor inducing conformational changes, activating the G-protein–guanosine diphosphate complex

A guanosine diphosphate-guanosine triphosphate exchange releases the alpha subunit of the G protein, which acts on other membrane-bound intermediaries called effectors

The effector is an enzyme that converts an inactive precursor molecule into an active scone messenger, which can diffuse through the cytoplasm and carry the signal beyond the cell membrane, inducing a cascade

39
Q

Endoplasmic Reticulum

A

A continuous system of membranes, intercommunicating channels and sacs which encloses a space called a cisterna in eukaryotic cells

In many places, the cytosolic side is covered by polyribosomes synthesizing protein molecules, which are then injected into the cisternae (rough)

40
Q

The principle function of the RER

A

To segregate proteins not destined for the cytosol.

Initial (core) glycosylation of glycoproteins

Synthesis of phospholipids

Assembly of multichain proteins

Post-translational modifications of newly formed polypeptides

41
Q

Functions of SER

A

Oxidation, conjugation and methylation processes employed by the liver to degrade certain hormones and neutralize noxious substances.

Synthesis of phospholipids for all cell membranes

42
Q

Function of the Golgi Complex

A

Completes post-translational modifications and packages and places an address on products that have been synthesized by the cell.

Condensing vacuoles bud from the Golgi cisternae, generating vesicles that will transport proteins to various sites

Glycosylation, sulfating, phosphorylation and limited proteolysis of proteins

Initiates packing, concentration, and storage of secretory products

43
Q

Lysosomes

A

Sites of intracellular digestion and turnover of cellular components.

Membrane-limited vesicles that contain a large variety of hydrolytic enzymes whose main function is intracytoplasmic digestion

44
Q

Lysosomal enzymes

A 
Acid phosphatase
Ribonuclease
Deoxyribonuclease
Proteases
Sulfatases
Lipases
beta-glucuronidase
45
Q

________ are synthesized and segregated in the ________ and subsequently transferred to the Golgi complex, where the ________ are modified and packaged as lysosomes.

A

Lysosomal enzymes
RER
enzymes

46
Q

Primary Lysosomes

A

Lysosomes that have not entered into a digestive event.

Fuse with phagosome and empty their hydrolytic enzymes into the vacuole, becoming a secondary lysosome

47
Q

Secondary Lysosome

A

When primary lysosomes fuse with phagosome and acidify the vacuole.

After digestion, nutrients diffuse through the lysosomal-limiting membrane and enter the cytosol.

48
Q

Residual bodies

A

Indigestible compounds that are retained within the vacuoles

In some long-lived cells, large quantities of residual bodies accumulate as lipofuscin.

49
Q

Melanosis coli

A

Disorder of pigmentation of the wall of the colon as a result of lipofuscin in macrophages.

Lipofuscin also seen in lymph node macrophages in wistar rats

50
Q

Autophagosomes

A

Secondary lysosomes that fused with membrane-enclosed organelles or portions of the lysosome during turnover of cytoplasmic organelles.

Cytoplasmic digestion by autophagosomes is enhanced in secretory cells that have accumulated excess secretory product.

Digested products of lysosomal hydrolysis are recycled by the cell to be reutilized by the cytoplasm.

51
Q

Proteasome

A

Multiple-protease complexes that digest proteins targeted for destruction by attachment to ubiqitin

52
Q

Function of Proteasome

A

Orchestrate protein degradation to remove excess enzyme an other proteins that become unnecessary to the cell after they perform their normal functions or incorrectly folded proteins

Degrades viral protein

Degrades individual proteins vs whole organelles like lysosomes

53
Q

Ubiquitin

A

Small protein found in all cells that targets proteins for degradation.

54
Q

Steps of Ubiquitin Degradation

A

A Ubiquitin molecule binds to a lysine residue in the protein to be degraded

Other Ubiquitin molecules attach to the first one

The complex is recognized by the regulatory particle that contains ATPase

The protein is unfolded by the ATPases using ATP as energy

The protein is translocated into the core particle, where it is broken into peptides of about 8 amino acids each

The peptides are transported into the cytosol where they are broken down to amino acids by cytosol enzymes

55
Q

Apoptosis

A

Programmed cell death

56
Q

Signs of an apoptotic cells

A

Compact, dark-stained nucleus (pyknotic nucleus)

Chromatin is cut into pieces by DNA nucleases (TUNL assay)

Blebs (cytoplasmic large vesicles) detach from the cell surface

Blebs remain within the plasma membrane so that it can be readily phagocytosed by macrophages

57
Q

Goblet cell

A

Mucus secreting cell of the gut and lung

Contains strongly hydrophilic glycoproteins called mucins

Secretory granules fill the extensive apical pole of the cell, with the nucleus at the base which is rich in RER and a well-developed Golgi complex

58
Q

Glycosyltransferases

A

Enzymes in goblet cells found in the RER and Golgi complex that add monosaccharides to core proteins to create mucins/glycoproteins

59
Q

Connective Tissue

A

Provides a matrix that connects and binds the cell and organs and ultimately gives support to the body

The major constituent being ECM (protein fibers and ground substance)

60
Q

Three classes of connective tissue components

A

Cells
Fibers
Ground substance

61
Q

Ground substance

A

Highly hydrophilic, viscous complex of anionic macromolecules (glycosaminoglycans & proteoglycans) and multiadhesive glycoproteins (laminin, fibronectin) that imparts strength and rigidity to the matrix by binding to receptor proteins (Integrins) on the surface of cells and to other matrix components

62
Q

The _________________ is the medium through which nutrients and metabolic wastes are exchanged between cells and their blood supply

A

Connective tissue matrix

Macrophage (3 decks)

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