Newbie at Flow cytometry so I need a lot of help - (Jun/12/2014 )

Hi Everyone,

I am not familiar with Flow cytometry but I need to run one to check the expression of ALK in my cell lines. I have a positive and a negative control cell line. I have also purchased the isotype control antibody. This antibody has not been optimized for FLOW but has been used for IHC so it is a novel attempt. Can anyone give/suggest a protocol that works and will be easy for me to follow? I will really appreciate all the help I can get.  I need to get this done as soon as possible.

Thanks a lot in advance

Is ALK expressed in the surface, cytoplasm or nucleus (or more than one location)? Asking to determine if you need a surface staining protocol or an intracellular one.

Tabaluga on Thu Jun 12 16:42:25 2014 said:

Is ALK expressed in the surface, cytoplasm or nucleus (or more than one location)? Asking to determine if you need a surface staining protocol or an intracellular one.

Thanks Tabaluga, ALK has both extracellular and transmembrane domain.

I assume your antibody targets the extracellular part, as is often the case with transmembrane proteins. Then you can do a surface staining. I forgot to ask one thing - is your AB and isotype ctrl directly labeled with a fluorochrome or not ?

Tabaluga on Thu Jun 12 16:54:31 2014 said:

I assume your antibody targets the extracellular part, as is often the case with transmembrane proteins. Then you can do a surface staining. I forgot to ask one thing - is your AB and isotype ctrl directly labeled with a fluorochrome or not ?

It is not directly labelled so I will be using a labelled secondary antibody.

Here I wrote you a general protocol like I used it for indirect FACS surface stainings; for FACS Buffer you can use something like PBS/1% BSA or PBS/5% FBS. You can try this one (of course there is a variety of other protocols available on the web as well). If anything is unclear, just ask.

Harvest cells, count them and resuspend a certain number (i.e. 10^6 cells/tube)

Wash in FACS Buffer (=resuspend, centrifuge and discard supernatant)

Incubate with primary antibody/isotype ctrl (diluted in FACS Buffer) at appropriate concentration for 1 hour at room temperature.

(You may need to titrate in order to find the best concentration, or for a start you can try out the manufacturer's recommendations or check if the antibody was already used in a publication and how it was used there. Same goes for time and temperature; do you have anything to go upon like a manufacturer's recommendation ? Also note that the isotype ctrl should be used in the same amount as the antibody - since concentrations may be different, you may need to calculate to really use the same amount.)

3x wash with FACS Buffer

Incubate with secondary antibody (diluted in FACS Buffer) at appropriate concentration (again, you may need to titrate and find the right conditions) at room temperature for 35 min in the dark

3x wash with FACS Buffer

Resuspend in FACS Buffer and measure; keep the samples cold and dark (e.g. in a closed ice box) and measure as soon as possible

Tabaluga on Sat Jun 14 11:37:42 2014 said:

Here I wrote you a general protocol like I used it for indirect FACS surface stainings; for FACS Buffer you can use something like PBS/1% BSA or PBS/5% FBS. You can try this one (of course there is a variety of other protocols available on the web as well). If anything is unclear, just ask.

 

Harvest cells, count them and resuspend a certain number (i.e. 10^6 cells/tube)

 

Wash in FACS Buffer (=resuspend, centrifuge and discard supernatant)

 

Incubate with primary antibody/isotype ctrl (diluted in FACS Buffer) at appropriate concentration for 1 hour at room temperature.

 

(You may need to titrate in order to find the best concentration, or for a start you can try out the manufacturer's recommendations or check if the antibody was already used in a publication and how it was used there. Same goes for time and temperature; do you have anything to go upon like a manufacturer's recommendation ? Also note that the isotype ctrl should be used in the same amount as the antibody - since concentrations may be different, you may need to calculate to really use the same amount.)

 

3x wash with FACS Buffer

 

Incubate with secondary antibody (diluted in FACS Buffer) at appropriate concentration (again, you may need to titrate and find the right conditions) at room temperature for 35 min in the dark

 

3x wash with FACS Buffer

 

Resuspend in FACS Buffer and measure; keep the samples cold and dark (e.g. in a closed ice box) and measure as soon as possible

Thanks a lot Tabaluga. The antibody has not been used for flow cytometry so there is no manufacturers recommendation for this application. For Immunohistochemistry it is recommended to be used at 1:250 dilution. Do I need to do replicate or it does not matter.

You should titrate the AB, so try with several dilutions. 1:250 could be one of them.
As for replicates, it is always recommendable but for your initial experiments (AB titration, adjusting the settings etc.) I'd say you can do without.