Flow Cytometry - part 2

Question 1

What can bead arrays detect?

Answer 1

Bead arrays for detection of proteins in solution:
Fluorescent beads coated with antibodies against molecule of interest
Mixture of beads with antibodies against different molecules

Question 2

What are the 3 steps to do intracellular staining (for cytokines for example)

Answer 2

1. Stimulation: stimulate the cell so that cytokines are produced
2. Transport block: block the transport of the cytokines in the cell
3. Fixation, permeabilization and staining

Question 3

What do you need for RNA flow cytometry?

Answer 3

Labeled complementary probe for mRNA

Question 4

How can we probe cellular functions with flow cytometry (proliferation)?

Answer 4

1. Cell cycle staining with DNA-binding dyes (for example DAPI)
2. BrdU or EdU incorporation (EdU and BrdU are thymidine analogs => in case one of them incorporated into DNA of proliferating cells: chemical reaction that covalently links dye to one of them)

Question 5

How can we probe cellular functions with flow cytometry (apoptosis)?

Answer 5

Annexin V: protein that binds to nuclear membrane
Propidium iodide (PI): DNA-binding dye
If PI detection then cell is in apoptosis (because nuclear membrane is open)

Question 6

How can you sort cells with flow cytometry?

Answer 6

Charging drops of interest: program the machine to charge (+/-) the target cells and fall into a tube

Question 7

What does a spectral flow cytometry allow?

Answer 7

Allows simultaneous detection of fluorochromes with very similar emission maximum => much more fluorochromes can be detected simultaneously