Question

Staining and Flow Cytometry

1. Procedure, controls and autofluorescence. Your ESC cultures are ready for a flow cytometry experiment. You wish to assess their undifferentiated status. You are going to do a double staining of two intracellular proteins, Oct 4 and Nanog.

    

1. For intracellular staining, you would need to…

Write the letter of the best answer in the blank (only one answer is correct):_____________

1. harvest the cells and incubate them with the fluorescent-conjugated antibodies.
2. harvest the cells, fix them, permeabilize their cell membranes and incubate them with the fluorescent-conjugated antibodies.
3. harvest the cells, fix them and incubate them with the fluorescent-conjugated antibodies.
4. harvest the cells, permeabilize their cell membranes and incubate them with the fluorescent-conjugated antibodies.

2. Your antibodies against the proteins of interests are:

• anti-Oct 4-FITC (IgG1) for green channel or FL1
• anti-Nanog-PE (IgG2a) for the red channel or FL2

Please explain which controls would you need for this experiment.

3. How would you deal with high autofluorescence in the red channel?

Write the letter of the best answer in the blank (only one answer is correct):_____________

1. Increase the voltage in the FL-1 channel.
2. Decrease the voltage in the FL-1 channel.
3. Decrease the voltage in the FL-2 channel .

4. Which control do you think is more appropriate to reduce autofluorescence in the red channel?

Write the letter of the best answer in the blank (only one answer is correct):_____________

1. Isotype control conjugated with PE.
2. Positive control with PE.
3. Unstained cells

Answer 1

1) For intracellular staining

Answer b) harvest the cells, fix them, permeabilize their cell membranes and incubate them with the fluorescent-conjugated antibodies.

Reason- Flow cytometry is a process for

2) Your antibodies against the proteins of interests are:

anti-Oct 4-FITC (IgG1) for green channel or FL1

anti-Nanog-PE (IgG2a) for the red channel or FL2

which controls would you need for this experiment.

Answer- Isotype controls are of great advantage. Isotype controls are used for identifying non-specific staining caused by primary antibodies. An isotype control consists non-specific characteristics of target antibody (primary). hence, could be used for estimating level of specific staining.

3) How would you deal with high autofluorescence in the red channel?

Answer- Decrease the voltage in the FL-2 channel, increasing voltage results into increasing the signal to background noise. since there is high red autoflorescence, therefore decreasing the voltage in FL-2 would decrease the autofloresence.

4) Which control do you think is more appropriate to reduce autofluorescence in the red channel?

Answer - Isotype control conjugated with PE.