Answers to Week 16 posting (For Li Ping and Ernest)

I understand that the answers I provide is quite long, but it's going to give you a clearer picture in some of the applications of flow cytometer, especially regarding my MP. Please don't give up on the 'long' post and read it =)

Before I start answering questions, I would like to touch on a few terms first (you could skip this part if you wish):

1) Fluorescence-based techniques
2) What is autofluorescence?
3) What is ‘true negative’?

Answers:

1) You should know that fluorescence-based techniques use fluorochromes in many different ways; one of the ways is to utilize the ability of dye to influx in the cells and ability of cells to efflux out the dye. Examples of such dyes are: Hoechst 33342 and Rhodamine.
These fluorochromes emit a certain ‘light’ wavelength after excitation from a light source (eg. Laser beam) that is detected by a detector in the Flow cytometer. Please take note that flow cytometer is mostly used to analyze live cells.
2) Autofluorescence is ‘weak’ autofluorescence emitted by cells not stained with the fluorochromes. Therefore, they are considered to be ‘false positive’ fluorescence.
3) As you will know, before the immunofluoresence staining of a cell, the cell should not express any fluorescence, right? So, ‘true negative’ is the sum of ‘negative’ and ‘false positive’ fluorescence.

To Li Ping’s Questions

Answers:

I believe you are confuse over the word ‘true negative’, when I mention about ‘true negative’, I meant the portion of cells in a heterogeneous cell population that are not stained positive. Only 'unstained' tube consist of 'negative' results as it is not stained at all. Other 'stained' tubes consist of both stained and unstained cells, so the data from 'unstained' tube is used to identify the cells that were not stained, something like a negative control.

And yes, it is correct to say that the value obtained will be used as benchmark, however, this benchmark is to identify the amount of ‘true negative’ population in other tubes. You must know that the cell sample we used is a heterogeneous population of cells and so, not all cells will be stained with the dye we used.

Different gates are used for different experiments. For my experiment, I used that series of gating that I mentioned before:

a. Scatter gate:
used to gate cluster of cells which our cells of interest is located and exclude all other ‘rubbish’ cells.
b. Side Scatter (SSC) gate:
used together with FSC for doublets discrimination and exclude all but single cells.
c. Forward Scatter (FSC) gate:
used together with SSC for doublets discrimination and exclude all but single cells.
d. Viability gate:
identify viable, living, cells. Cells not stained by Propidium Iodide (PI), one of the viability dyes that could be used, are live cells.
e. Live gate:
exclude ‘rubbish’ cells, like red blood cells, and non-viable cells that are not successfully excluded by previous gates. Also exclude other cells that are not of interest.
f. SP gate:
compare “Hoechst 33342 only” against “Hoechst 33342 + Verapamil” tubes to identify and gate SP cells.

Note: Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.

To Ernest Questions

1) Yes, you can interpret it as that way. ‘Unstained’ tube contains cells that are not stained. This is used to compare with other ‘stained’ tubes, which consist of both stained and unstained cells in its pool of heterogeneous cell population. It helps to identify cells that are not stained from the rest of the stained cell population. Something like a negative control.

2) ‘PI only’ stands for the tube that is only stained by propidium iodide, which is one of the viability dyes that could be used. Cells not stained by PI are live cells.

3) You can add as many dyes as you can, however, people usually 2 different dyes plus a viability dye at most. Also, it doesn’t make sense to add too many dyes as there will be complications like spectrum overlap of fluorescence that spills into other ‘light’ channels and gives false positive results.
(For example only: Imagine having PE dye fluorescent spills over into FITC dye channel and gives a false positive result in the FITC dye channel as the fluorescence detected in FITC channel is actually from the PE dye)

4) Please refer to above answers to Liping’s questions.

5) Please do not confuse my experiment to detect SP cells from an unrelated experiment of DNA analysis that also uses Hoechst 33342 dye. SP cells are known to actively efflux out drugs and dyes like Hoechst 33342, which is why Hoechst 33342 is used in our experiment design. And we compare Hoechst red to Hoechst blue channels to observe our results.

As I mentioned in answering Liping’s question:

“Blockers, like verapamil, inhibit ABC transporters that give a ‘tail-like’ cell profile of SP cells that are low Hoechst blue and red, on the plot graph.
Hoechst emits a wavelength that can be detected at two different channels with two different detectors.” (Quoted)

6) Refer to above, answer to your question 5.

I really hope both of you understand more about my SP experiment. If you have any more doubts, feel free to ask again.

Also, to understand more about my SP experiment, please read my 3rd posting of “Double posting! (2nd part: SP Profiling in Stem Cells)”, before asking me any related questions.

Many thanks
Quan Jun