How do you make your media?

Dear Lillian, the medium I used for lymphocyte culture is RPMI 1640 medium (containing L glutamine), 10% FBS, 1mM sodium pyruvate, 10mM non-essential amino acids, 100U/ml penicillin, 100ug/ml streptomycin, 50 uM 2-mercaptoethanol.

Dear Mr John, thank you for your suggestion! Definitely I will try ! Just one question, could adding IL-12 make T cell skew toward Th1 phenotype ? 

Dear Mr John,

I want to transfer both Th1, Th17  in stead of a specific phenotye, what should I do ?

Dear Mr John, 

I appreciate all your comments very much ! Your suggestions help me understand this  model better and design experiments. I hope we can discuss more in the future.

Best Regards,

Dear Minh,

If your just want to induce passive EAE, you may do as follow:1. immunized 2-5 mice at hind footpads with 200 ug pMOG35-55 with CFA (containing 300 ug M.t.), and on Day9, sacrifice mice to isolate draining lymphoid nodes to make single cells suspension.  For good immunization, from 1 mouse, one can obtain around 30 millions cells.  You may pool cells from all mice immunized and use them for culture (next step).  2. Culture the cells with RPMI1640 (with L-glutamine) supplemented with 0.5% mice sera (may obtain from naïve B6 mice), 100U/ml penicillin, 100ug/ml streptomycin, 50 uM 2-mercaptoethanol. You should use the density of 4-5 million cell per ml in 24-well plate (or 16 million cells per well (4 ml) if you use 6-well plate), and add pMOG35-55 at 10ug/ml (immediately to about 1 h after you set up the plates).  In the lymphoid cells you obtain from immunized mice, the pMOG35-55-specific T cells were greatly expanded and skewed to Th1 and Th2 (you may check the lymphoid cells by intracellular staining IFNgamma- and IL-17A-secreting CD4 cells. However, they also contain a lot of activated, non-MOG35-55-specific T cell, B cells, etc.  By using mouse sera and not using rIL-2 at this initial culture, you can more specifically expanded the MOG-specific T cells. 3. (Critical!) Check your culture frequently (twice a day, in the morning and in the evening), usually after 36-48 h after you set up you culture, you should see vigorous growth of T cells (they form big clumps), and you should do SPLITTING:  Add 2-5 ml RPMI1640 (with L-glutamine) supplemented with 10% FBS, 100U/ml penicillin, 100ug/ml streptomycin, 50 uM 2-mercaptoethanol, and rmIL-2, mix the cells and then put cells suspension to new plates (use 6-well plate to save your operation, at 4 ml per well).  This splitting is critical to avoid culture to become overcrowded which results in cell death from overcrowded and lacking of neutrition.  You should keep the original wells and feed them with same medium (they still contain a lot of desired cells).  4. After splitting, keep checking culture, and do splitting again if needed.  5. By day 3 (or 4), you can harvest your culture for adoptive transfer of EAE, you may also take a little cells to do FACS to analyze their immune-phenotypes.

With such care, you should obtain enough cell for inducing passive EAE.     

Dear Ming,

Thank you for your detail comment!  Would you explain for me a little more about your protocol?

1. Why do you add rmIL-2 into the medium when splitting cells?

2.The second medium contains L-glutamine, FBS, antibiotic, 2ME but not the MOG, is it OK ? I am afraid that by doing that I will decrease the concentration of MOG in the initial medium (from 10ug/ml to 5g/ml->> I think it is quite low)

3. Approximately after 3 days of culture, how about the concentration of living cells can you obtain from 4-5 million cell per ml at day 0?

Dear Minh,

1. IL-2 can promote the survive and proliferation of CD4+ T cells and other immune cells (such as NK cells).  Upon activation, CD4+ T cells can also secrete IL-2, but they also consume IL-2 quickly. After you set up the initial culture with cells, medium without IL-2, and the antigen pMOG35-55, pMOG35-55-specific T cells will be (RE-)stimulated by APC loaded with pMOG35-55.  By not adding IL-2 at this stage, you can reduce the non-specific proliferation of other immune cells.  After 36-48 h when you can observe vigorous proliferation of T cells (you may see big clumps), IL-2 that you are going to add in the splitting medium can promote further the proliferation of antigen-specific CD4+ T cells.  As the end result, you will obtain much more pMOG35-55 specific CD4+ T cells for adoptive transfer of EAE.

2. No need to worry for this.  pMOG35-55 has to be taken up by APC, loaded to MHC-II, and presented to pMOG35-55-specific CD4+ T cells, so that it can initiate the activation and proliferation of pMOG35-55-specific CD4+ T cells.  After antigen-specific T cells enter the proliferation cycle, they can continue to proliferate in a manner  independent of pMOG35-55/MHC II complex on APC.

3. Under good care conditions, you may obtain > 4 millions of activated CD4+ T cells on day 3, starting from 1 well culture (4 millions of lymphoid cells from lymph nodes).  This yield make the adoptive transfer of EAE feasible. 

4. One more thing: The quality of immunization can also affect the yield of pMOG35-55-specific CD4+ T cells.   Immunization, especially good immunization, can greatly increase the frequency of pMOG35-55-specific CD4+ T cells in lymphoid cells, and you may obtain much more of pMOG35-55-specific CD4+ T cells at the end of culture. 

Hope you can succeed soon with your experiments.

Ming-Chao

Dear Ming,

Thank you so much for your really nice explanations ! I have learnt a lot of interesting things from your comment! By the way

1) What do you mean "good immunization" ? Does it relate to the amount of peptide or the way you inject it? I usually prepare for each mice 200ul of an emulsion containing 100 ug of MOG35-55 and 500 ug of Mycobacterium tuberculosis in CFA. Then the emulsion is subcutaneously injected over 2 sites: 100 ul along the midline of the back between the shoulders and 100ul on the midline of the lower back.

2) Is it necessary to inject the Pertussis toxins to make a "good immunization" ? I read somewhere that the toxins can affect the immune system.  

3) Why do you use only lymph node instead of the combination of spleen and lymph node? And for the lymph node, I collect them from the inguinal and axillary sites. 

Best Regard,

1. For isolating lymphoid cells for passive EAE, injecting at two hind footpads and then harvesting the draining lymph nods is much better that injecting at s.c. flank and harvesting spleen and lymph nodes (The latter one is commonly used for inducing active EAE).  The cells from draining lymph nods are much "pure" than those from splenocytes.  From good immunized mice,  you can obtain very big draining lymph nods on day 9-10 and harvest >30 millions of lymph nod cells from one mouse.  You can make emulsion containing pMOG35-55 at 1-2mg/ml and M.t. 1.5-2.5 mg/ml, and inject to each hind footpad with 50 ul.  The quality of emulsion and injection can affect the yield of the pMOG35-55-specific T cells. 

2. No need to inject PT.  When mice are used for induction of active EAE, some people inject PT (to break the blood-brain barrier etc).  For your purpose here, it is no needed at all to inject PT (Besides, PT is highly toxic to human, and also expensive).

3. Lymph nod cells, most of them  come from immune response, are much "pure" than  splenocytes. From splenocytes, there are much more "contamination" of fibroblast-like cells, and these cells can proliferate and almost cover the whole bottom of wells after 2-3 days' culture. (You can compare this by culturing lymph nod cells and splenocytes  separately, and then you can how differently these cells are).  

Dear Ming,

Thank you for your quick response! I really appreciate all your comments. I think you should publish a paper for making adoptive EAE! I guess it will be cited in many papers.

Best wishes for you!