Research: Oxidase activity of neutrophilic granulocytes

Question 1

Neutrophiles are essential in innate immune response and can phagocytose pathogens.
> to recognize them, the pathogens need to be (1)
> This process is called (2)
> After phagocytosis, (3)

Answer 1

(1) Coated with opsonins IgG and C3bi
(2) Opsonization
(3) The phagosome contents will be degraded with the use of NADPH-oxidase which forms toxic oxygen metabolites to destroy the pathogen

Question 2

The opsonins C3b and IgG are bound by … of the neutrophilic granulocytes

Answer 2

CR3 and Fc receptor

Question 3

What happens after opsonin binding

Answer 3

Engulfing of the pathogen, phagosome fuses with lysosome and breakdown of pathogen
> Through receptor activation, the PKC route gets activated in neutrophil
> NADPH oxidase is localized inside phagolysosome
» after activation, the NADPH-oxidase complex is formed at the inner membrane of the phagolysosome
» release superoxide, hydrogen peroxide etc will be formed by reduction of oxygen (eventually to hypochlorus by MPO)
» NADPH oxidation in cytosol while O2 reduction inside phagolysosome

Question 4

Nitro-blue tetrazolium in phagocytosis assay

Answer 4

NBT is converted to blue colored Formazan in a redox reaction with superoxide (O2-), forming O2 as well
> measure oxidase activity

Question 5

Positive control for oxidase activity

Answer 5

Through addition of PMA and no zymogen particles for phagocytosis
> PMA activates PKC and the NADPH-oxidase complex without the binding of opsonins and the receptor induced pathway

Question 6

How is phagocytosis and NADPH-oxidase activity in assay

Answer 6

Under microscope
> particles in vesicles: phagocytosis
> blue color: NADPH activity through opsonin receptor induced route or PMA positive control

Question 7

What would happen to phagocytosis activity and oxidase activity in the assay when:
You forget resuspending the cells before dividing them over 4 tubes

Answer 7

Phagocytosis: higher/lower
Oxidase: higher/lower

Question 8

What would happen to phagocytosis activity and oxidase activity in the assay when
You forget to resuspend the zymosan particles

Answer 8

Phagocytosis: higher/lower
Oxidase: higher/lower

Question 9

What would happen to phagocytosis activity and oxidase activity in the assay when
You forget to add NBT solution

Answer 9

Phagocytosis: equal
Oxidase: lower

Question 10

What would happen to phagocytosis activity and oxidase activity in the assay when
You incubate on ice instead of 37 degrees

Answer 10

Phagocytosis: lower
Oxidase: lower
» phagocytosis is an active process that takes place at optimal temperature of 37 degrees, longer incubation at this temperature will increase phagocytosis activity. On ice, this process is stopped.

Question 11

What would happen to phagocytosis activity and oxidase activity in the assay when
You incubate for 1 hour instead of 10 minutes

Answer 11

Phagocytosis: higher
Oxidase: higher
» phagocytosis is an active process that takes place at optimal temperature of 37 degrees, longer incubation at this temperature will increase phagocytosis activity

Question 12

After the ficoll density gradient centrifugation, the pellet contains neutrophilic granulocytes and erythrocytes. How are neutrophils isolated?

Answer 12

Add lysis buffer to isolate the neutrophlic granulocytes and lyse erythrocytes > hemolysis

Question 13

Lysis buffer with NH4Cl (ammonium chloride)
> lysis buffer can contain different compounds, why
> how does it work

Answer 13

> depending on the cells you want to lyse and which ones you want to isolate
membranes of erythrocytes contain anion exchangers which can exchange (hydrogen carbonate) HCO3- outside for Cl- (chloride) to the inside.
when adding lysis buffer, chloride is driven into the cell due to ion gradient, causing hydrogen carbonate to be driven out of the cell by the anion exchanger
Carbonic anhydrase can convert HCO3- to CO2 + H2O and vice versa inside erythrocyte
as compensation of decrease in HCO3-, carbonic anhydrase uses CO2+H2O (equilibrium of reaction towards HCO3-)
To compensate for water depletion, water will be taken up by the cell, causing the cell to burst open or lyse

Question 14

Why can neutrophilic granulocytes be isolated with the lysis buffer from erythrocytes

Answer 14

Only erythrocytes are lysed by lysis buffer
> after lysis incubation, centrifugation and derive pellet (white)
> supernatant turns red: hemoglobin released from erythrocytes when lysis
> lysis has to be repeated couple of times for complete erythrocyte lysis and neutrophilic granulocyte purification

Question 15

Why are neutrophilic not affected by lysis buffer

Answer 15

Process is slower in neutrophilic granulocytes, but they are affected!
> also can take up ammonium chloride with anion exchanger and hydrogen carbonate is exported out
> but it does not have carbonic anhydrase and water depletion does not occur as quickly as in erythrocytes
> chloride uptake does change osmolarity in the cell, and lysis can occur over time: water uptake still.

> > lysis step is time-dependent: erythrocytes lyse first and incubation should not take too long

Question 16

Why is the hemolysis step in neutrophil purification taken place on ice

Answer 16

Because the erythrocytes lyse earlier than neutrophils, but to increase the gap and prevent lysis of neutrophils, the lysis is slowed down by performing it on ice.

Question 17

Why is HEPES medium chosen after purification of neutrophilic granulocytes?

Answer 17

To optimialize pH for an enzyme, likely NADPH oxidase in the phagocytosis assay

Question 18

EDTA in lysis buffer

Answer 18

Metal chelator, to prevent aggregation and possible sinking of RBC elements to pellet

Question 19

KHCO3 in lysis buffer

Answer 19

Prevent excessive swelling or damage to other cells while lysis, by maintaining osmotic balance during the lysis process with potassium K+ ions
> and to stabilize pH for carbonic anhydrase in the lysis

Question 20

Why will there be washed with PBS after incubation with lysis buffer two times?

Answer 20

To remove the lysis buffer

Question 21

Why are zymosan particles used in a phagocytosis assay

Answer 21

Zymosan particles are yeast cell walls, which are suitable for testing phagocytosis since they can be opsonized with antibodies and complement factors

Question 22

The patient has chronic infections, therefore neutrophilic granulocytes are tested for:

Answer 22

Phagocytosis, NADPH oxidase activity and migration