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Neuro Lab Flashcards

1
Q

What is GlcNAc?

A

This is an enzyme that drives formation of complex N-glycosylation through Mgat5

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2
Q

What enzyme drives formation of complex N-glycosylation, and how does it do it?

A

GlcNAc, which works through Mgat5.

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3
Q

What is Mgat5 antagonized by?

A

Mgat3.

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4
Q

What is Mgat3 antagonized by?

A

Mgat5.

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5
Q

How can surface glycoproteins be tagged?

A

By oxidizing the sugars present and labelling with biocytin hydrazide.

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6
Q

What does biocytin hydrazide do?

A

This chemical labels oxidized sugars.

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7
Q

What enzyme cleaves some glycosydic linkages?

A

PNGase F.

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8
Q

What does PNGase F do?

A

This enzyme cleaves some glycosidic linkages.

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9
Q

What is the MCAO stroke model?

A

This stands for Middle Cerebral Artery Occlusion, and is used to replicate the effects of an ischemic stroke.

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10
Q

What is the integrin subunit that differs between neurogenic and astrogenic NPSCs?

A

Integrin subunit alpha-5, which binds to the ECM molecules fibronectin and vitronectin.

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11
Q

What is one thing that controls residence time of receptors on the plasma membrane?

A

The N-glycan content of that receptor.

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12
Q

Describe the process for immunostaining.

A
  1. Clean out the wells you are using with 1X PBS three times, waiting 5 minutes after each wash, letting the PBS soak.
  2. Prepare the serum albumin solution
    5% V/V, so 5g per 100mL of PBS
    mix or vortex until serum albumin fully dissolved
    filter with 0.45um filter, using appropriately sized syringe, plunging directly into new vial.
    for extended use, use ice. Otherwise store in walk-in freezer
  3. Add BSA solution with Pasteur pipette to cell wells and wait one hour.
  4. Make antibody solution in 1% BSA, typically 1:100 antibody dilution.
  5. Add antibody solution in droplets to parafilm-secured glass plate, then place the cell slides onto the droplets.
  6. Let sit overnight in walk-in freezer
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13
Q

Describe the process of dissociating mouse neural stem cells.

A
  1. Remove cell suspension with micropipette and place in centrifuge tube
  2. Centrifuge at 1.1k RPM for 5 minutes
  3. Extract liquid with Pasteur pipette hooked up to safety cabinet vacuum.
  4. Add 150uL of “Solution A” to the cells, mixing with the pipette.
  5. Add 38uL of “Solution B” to the cells, mixing with the pipette. Let sit for 8 minutes, mixing again at 5 minutes remaining, 1 minute, and 0 minutes.
  6. Add 12uL of “Solution C” to the cells, mixing with the pipette.
  7. Add 800uL of medium(+)
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14
Q

Describe the process for counting mouse neural stem cells.

A
  1. Extract 10uL of suspension from well or test tube, and mix with 10uL of Trypan BLUE.
  2. Count the cells in each quadrant, where each quadrant contains 0.1uL of suspension, counting at least 100 cells.
  3. Find the total number of cells by using this equation:
    #Cells / #Quadrants * 1 quadrant/0.1uL * 1000uL/1mL
    Simplified: Cells / Quadrants * 10,000 = cells per mL.
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15
Q

Describe the process of seeding a new mouse neural stem cell well.

A
  1. Add at least 300,000 cells per well in 3mL total of medium(+).
  2. Label wells and place in incubator, check on the cells daily.
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16
Q

What does ruthenium red bind to?

A

Sialic acid and other acidic molecules.

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17
Q

What dye binds to sialic acid and other acidic molecules?

A

Ruthenium red.

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18
Q

What is the glycocalyx?

A

The fuzz-like coat of carbohydrate moieties surrounding a cell.

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19
Q

What is the fuzz-like coat of carbohydrate moieties surrounding a cell called?

A

The glycocalyx.

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20
Q

What is a moiety?

A

A functional group or certain section of a molecule.

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21
Q

What does prolonged unfolded protein response lead to?

A

Programmed cell death.

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22
Q

What is the most biosynthetically proximal enzyme for mature N-glycans?

A

GlcNAc-T1, which is encoded by Mgat1.

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23
Q

What does the molecule lectin have selectivity for?

A

This molecule has binding selectivity for complex N-glycans.

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24
Q

What are some of the antibodies we use for identifying neurons?

A

MAP2, which is a dentdritic marker, and TUJ1/B-3 tubulin, which stains both dendrites and axons, as well as the soma, GABA, which stains the neurotransmitter, Glutamate, which stains the neurotransmitter, neurogenin, which labels neuron progenitors, and DCX, which works well on human stem cells, and stains the primitive cytoskeleton of newly developed neurons.

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25
Q

What are some of the primary antibodies we use for identifying astrocytes?

A

GFAP, which stains the glial cell cytoskeleton, and S100-B, which is a Ca2+ transporter specific to glial cells, as well as SOX-9, which is a transcription factor for astrogenesis.

26
Q

What are some of the primary antibodies we use for identifying oligodentdrocytes?

A

O4, which stains the a glycosphingolipid on the cell membrane, OLIG-2, which is an oligodentrodyte progenitor marker, and OLIG-4, which is used slightly further along than OLIG-2.

27
Q

What are some of the primary antibodies we use for identifying neural stem cells?

A

Nestin, which stains the cytoskeleton of neural stem cells, and SOX-2, which is a highly expressed transcription factor in neural stem cells.

28
Q

What are some of the primary antibodies we use for identifying proliferating cells?

A

Phosphohistone H3, which is a histone responsible for chromatin condensation, Ki67, which is constitutively expressed when cells are not in GO (but doesn’t work well in mouse cells), and EDU, which stains all newly-synthesized DNA, and is used in pulse-chase experiments.

29
Q

What is the enzyme mannosidase II?

A

It is an enzyme involved in N-linked glycan processing, inhibited by swainsonine.

30
Q

What is swainsonine?

A

A small molecula that inhibits mannosidase II.

31
Q

How are astrocyte progenitors enriched?

A

By using high-capacitance low-frequency DEP sorting, around 0-100kHz, potentially 0-50kHz.

32
Q

How are neuron progenitors enriched?

A

By using low-capacitance high-frequency DEP sorting, around 300-400kHz, potentially 350-400kHz.

33
Q

What are some man1 inhibitors?

A

The small-molecule inhibitors DMJ and Kifu (Kifunensine) both work to inhibit this enzyme.

34
Q

What does DMJ do?

A

This small molecule is a man1 inhibitor.

35
Q

What does Kifu (Kifunensine) do?

A

The small molecule is a man1 inhibitor.

36
Q

What are some man2 inhibitors?

A

SW and Mannostatin A are both inhibitors of this enzyme?

37
Q

What does SW do?

A

This molecule is a man2 inhibitor.

38
Q

What does Mannostatin A do?

A

This molecule is a man2 inhibitor.

39
Q

What does ICC stand for?

A

Immunocytochemistry.

40
Q

What is shRNA?

A

Small hairpin RNA, and it is used for silencing certain genes being expressed.

41
Q

How are cell-surface N-glycans detected?

A

With fluorescently tagged lectins

42
Q

Describe the process of culturing human neural stem cells.

A

X. Change 50% of their media each day by extracting with a pipette and then inserting a new amount of solution with a pipette.

43
Q

How can you track a cell through division?

A

By staining the cell with Cell Tracker Orange, which is detectable even after 4 rounds of cell division.

44
Q

What is Cell Tracker Orange used for?

A

It is used for tracking specific cells

45
Q

Describe the process of making N-feed media.

A

(Makes 25mL)

  1. Add 24.5mL MEM, 250uL B27, 25uL NGF, and 500uL Horse serum. Pipette each of these up and down before adding to main tube, then add to main tube.
  2. Mix tube by gently inversion repeatedly
  3. Filter the contents of the tube through a 0.22um filter. If making >50mL, use a vacuum filter, but if using
46
Q

Describe the process of making 1st-feed media.

A

(Makes 25mL)

  1. Add 235mL N-feed, 1.25mL fetal calf serum, and 250uL penicillin/streptomycin. Pipette each of these up and down before adding to main tube.
  2. Gently invert repeatedly to mix.
  3. Run through 0.22um filter and store media at 4 degrees C. Media stays good for up to a week.
47
Q

What binds to sugars in the ECM?

A

Lectin (also fibronectin).

48
Q

What do lectin and fibronectin bind to?

A

Sugars in the extracellular matrix, or glycosylation patterns extending from the plasma membrane.

49
Q

What protein is used to direct neural stem cell process formation?

A

NCAM, and especially when it is associated with polysialic acid.

50
Q

What is vecta-shield used for?

A

It removes reactive oxygen species created by fluorescing samples.

51
Q

How do you ensure samples stay fluorescent for as long as possible?

A

Place them in a solution of vecta-shield.

52
Q

Does size of cells correlate with passage number?

A

No.

53
Q

What is confocal microscopy used for?

A

Visualizing slices of a 3-D object and reconstructing it.

54
Q

How do you coat coverslips with laminin?

A
  1. Wash the wells with 1:50 diluted PDL (poly-D lysine) dissolved in MQ (milliQ water), wait 5 minutes.
  2. Wash the wells with 1:50 diluted laminin in MEM.
  3. Let sit for 4 hours to 4 days in an incubator at 37C.
  4. Wash with MEM, wait 5 minutes.
55
Q

How do you coat wells with fibronectin? Why?

A
  1. Wash the wells with 1:100 diluted fibronectin in MEM.
  2. Incubate for 4 hours to 4 days in 37C.
  3. Wash the wells with MEM for 5 minutes.
    To allow human neural stem cells to grow.
56
Q

What does ICC stand for?

A

Immunocytochemistry.

57
Q

What is DEP buffer made out of?

A

8.5% sucrose, 0.3% glucose, and adjusted to 110uS/cm conductivity with RPMI or KCl

58
Q

What does EGF stand for?

A

Epidermal growth factor.

59
Q

What does FGF stand for?

A

Fibroblast growth factor.

60
Q

What does PBS stand for?

A

Phosphate-buffered saline, which is a salt solution that is isotonic to the human body and frequently used in cell culture.

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