I need to titrate mouse CD107a antibody to be used for my future CD8+ T cell degranulation assays. After checking the literature, it is recommended to stimulate the cells with some antigen and add the CD107a antibody to the culture at the beginning of the stimulation. So, the strategy I will follow is :
1. Stimulate the CD107a stained splenocytes using anti-CD3/CD28 (plate bound) for total 5 hrs.
2. Add 1uM Monensin to the cultured cells for the last four hrs of incubation.
3. Harvest cells, wash and surface stain with anti-CD3 and anti-CD8 antibodies.
4. Fix and permeabilize the cells and acquire by Flow cytometer.
I was wondering if this is the right protocol to follow?
Also, is it fine to stain the splenocytes with anti-CD107a in complete RPMI medium as you need to stain the cells with this antibody in the beginning of the culture stimulation?
Is it necessary to fix and permeable the cells for checking the expression of CD107a antibody?
Please suggest.
Hi Saptha and Tabasum,
The idea behind your test is to measure the degranulation of your CD8 T cells post-stimulation. this can be measured in different ways, one of which is the arrival of trans-membrane granules marker (like CD107a/Lamp-1) to the cell surface. In cells not degranulating, this marker is strictly intra-cellular, however, when granules fuse with the cell-membrane to empty their content in the outside milieu, you'll be able to detect the markers of these intracellular granules on the cell surface w/o the need for permeabilization. Permeabilizing the cells will show you that any cell (activated or not) contains intracellular CD107a.
In this situation I wouldn't recommend using monensin as soon as you start your stimulation. I assume you would want to block the CD107a at the cell surface by using it 5hrs post activation, and this is ok, but not necessary, as you could stop intracellular trafficking by incubating on ice or at 4°C and staining right away for CD107a presence at the cell surface and going to the cytometer. Make sure you keep your cells at 4°C all the time until being acquired.
Antibody staining in complete medium works very well. Just avoid using avidin/Streptavidin ways of detection as media and serum contain some biotin and could interfere with the detection of your primary antibodies (that is if you are using an indirect system of detection)
Good luck!
I am not working with splenocytes rather with human T cells, but as far as i know you have to adjust the protocol to your desired output. That means, there is no "best" protocol and it can change with respect to your future experiments.
Before stimulation, add anti-CD107a. The maximum volume (cells + medium) of your well should not exceed 100µl to ensure staining as well as reproducibility.
I only can suggest the use of PMA and Ionomycin for stimulation (4h is enough) and Monensin is added right away (Saving time and steps). In addition, you dont need to fix and permeablize the cells.
Hope you will find your best-suited protocol.
Best regards
Thanks Julian. I will follow your suggestions and try to make the optimized protocol.
Hi Saptha and Tabasum,
The idea behind your test is to measure the degranulation of your CD8 T cells post-stimulation. this can be measured in different ways, one of which is the arrival of trans-membrane granules marker (like CD107a/Lamp-1) to the cell surface. In cells not degranulating, this marker is strictly intra-cellular, however, when granules fuse with the cell-membrane to empty their content in the outside milieu, you'll be able to detect the markers of these intracellular granules on the cell surface w/o the need for permeabilization. Permeabilizing the cells will show you that any cell (activated or not) contains intracellular CD107a.
In this situation I wouldn't recommend using monensin as soon as you start your stimulation. I assume you would want to block the CD107a at the cell surface by using it 5hrs post activation, and this is ok, but not necessary, as you could stop intracellular trafficking by incubating on ice or at 4°C and staining right away for CD107a presence at the cell surface and going to the cytometer. Make sure you keep your cells at 4°C all the time until being acquired.
Antibody staining in complete medium works very well. Just avoid using avidin/Streptavidin ways of detection as media and serum contain some biotin and could interfere with the detection of your primary antibodies (that is if you are using an indirect system of detection)
Good luck!
I have a question: if a blocker is necessary before adding the Ab anti-CD107a in culture medium to prevent the unspecific binding?
I have tried adding the CD107a staining antibody to the cell culture medium (human T cells with PMA/Ionomycin ) before incubation as well as surface staining after incubation. Both times monensin was added so I'm assuming this blocks the trafficking of the molecule back into the cell once it has reached the surface. When stain is added before incubation, % +ve is about 5% higher than when staining is done after incubation. Is this due to non specific binding? or is there still some trafficking going on that is not stopped by the monensin. due to the nature of the assay I cannot keep the cells on ice. Any input would be appreciated.
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