Can somebody suggest which murine cells should be used for titrating CD107 (LAMP1) antibodies for Flow Cytometry? My idea is that we can use irradiated splenocytes as APCs and stimulate these cells with the cytokine like IFN-gamma for titrating this antibody. Will it work? Please suggest.
Surface CD107 is detectable on activated CD8 T cells (because they degranulate and exposed CD107 on their surface). You could therefore stimulate mouse T cells (from the spleen) with anti-CD3 (or antigen if you have access to antigen-specific T cells) and then detect CD107 (see PMID 14580882)
This first answer sounds like a very elegant method. Alternatively, you might try to permeabilize cells with 0.02% saponin, stain for intracellular lamp-1, everything strictly in the cold. Depending on you antibody, you may also consider fixation with 1-4 % PFA in addition to permeabilization to stabilize the cells.
You should always try to titrate your antibody on the same type of cell your experiment will use. Assuming you will be looking at T cells, as Denis said stimulate isolated T cells with anti-CD3 or CD3/CD28 beads or PHA or SEB and then detect CD107. If you do not use isolated T cells be sure to use dumps for CD14 CD19 and the like to decrease your background.
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.
Fix/Perm is NOT required or advised for the detection of CD107. It is important that you understand what the CD107 assay is doing. It is not a normal surface molecule or intracellular molecule. CD107a/b (LAMP1/2) are molecules expressed inside granule lysosome membranes. When a T cell or NK cell degranulates, as the membrane fuses with the cell membrane to release its contents outside the cell. Momentarily, CD107 is therefore expressed on the surface, however, it will quickly be recycled by the cell. This is why you have added anti CD107 antibodies to the cell culture, to catch and detect these molecules. Monensin is required so that when the molecule, along with the antibody and fluorescent conjugate, is recycled back into the cell, the fluorochrome is not lysed and digested by the cell. Additionally, complete RPMI is fine for this assay. Please again read the publication mentioned above by Denis, PMID 14580882.
Thanks Colt for the important suggestions and making things more clearer. I really appreciate this.
Sure! It's definitely a different type of stain, so let me know if you have any other questions.
Hi Colt,
I was successful in checking the degranulation of CD3/CD28 stimulated CD8+ T cells from mice spleen using the CD107a antibody. I added this antibody to the cells at the beginning of the stimulated culture and used two conditions for Monensin addition (Monensin added right at the beginning of culture and after 1 hr of incubation followed by 4 more hours of incubation (Total five hrs). I observed that adding Monensin right at the start of stimulation culture works better. I then proceeded for checking the degranulaton of CD8+ T cells along with intracellular IFN-g production which is my main aim. I followed the same protocol, only with the addition of intracellular staining for IFN-g and found that even in unstimulated control there seems to be CD107 expression even on CD4 T cells and I don't see any IFN-g release in any of the conditions.
I am attaching my FACS panel so that you get the ides what actually is going on.
Please let me know if you have any suggestions how to troubleshoot IFN-g part.
What is the maximum time naive CD8+T cells from mice spleen take to get activated using PMA/Ionomycin or anti-CD3/CD28 stimulation?
Actually I want to measure CD107a and IFN-gamma expression from the naive CD8+T cells from mice spleen which is a kind of trial experiment before I go for the actual experiment where I need to infect mice with specific antigen to check these. I was wondering how long should I stimulate naive mice splenocytes with PMA/Ionomycin or anti-CD3/CD28 to check the simultaneous expression of surface CD107a and intracellular IFN-g? I know that we can check the CD107a expression on anti-CD3/CD28 stimulated CD8+ T cells in 5 hrs only. However, this strategy did not work for me for checking the IFN-g expression.
Please suggest.
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