Dear flowers, First of all, I would like to thank all those who responded to my question about detection of intracellular IL-4. For those of you who are interested, I have made a summary of the replies. From the answers I got, it seems I am missing the peak of IL-4 production, possibly combined with a problem of sensitivity. Possible solutions may be (i) looking after a few hours instead of overnight or (ii) adding Brefeldin A or monensin during the overnight culture. The full text of my question and the answers follows below: * My original question was: Could anyone of you tell me what are the best conditions (mainly time of restimulation, temperature of incubation, etc) to detect intracellular IL-4 in splenocytes in response to mitogen or antigen restimulation? We are working with splenocytes from parasite-infected animals. In ELISA we can detect IFN-gamma and IL-4 in the supernate when these cells are restimulated in vitro with mitogens such as ConA. However, whereas we can detect IFN-gamma intracellularly by flowcytometry after overnight incubation with ConA, we do not detect IL-4. After the overnight incubation with ConA, we are treating the cells for 5 h with Brefeldin A before the FACS staining. We are using Pharmingen's R-PE-conjugated anti-mouse IL-4 antibody (clone 11B11) at 0.2 microgram/one million cells and we are staining paraformaldehyde-fixed and saponin-permeabilized cells for 30 minutes at 4°C in the dark. *Jelia from Pharmingen replied: First question: What is the amount of IL-4 you are detecting in the sn ? My guess is the level is in the pico gram range. To determine by flow cytometry you need close to ng levels of cytokines. Next you might want to put the Brefeldin-A in the culture when the culture is initiated. You can keep the transport inhibitor in there overnight. Yes, you will get some cell death, but they can be gated out with a light scatter profile. *Joost Schuitemaker from UVA in the Netherlands replied: Although we are only working with human material, maybe the following could help you. Adding BrefA after an overnight stimulation to detect IL-4 is useless. The IL-4 will then already be in the supernate. You can check this once as a control. However, you can add the BrefA overnight without problems. My suggestion would be: I. Stimulation with PMA (10 ng/ml) / ionomycin (1 microgram/ml) during 6 h, of which the last 5 h in the presence of 10 microgram/ml BrefA. II. Stimulation with Con A O/N of which 1 h without BrefA. As maximum time, I would use 16-20 h. III. Should you be interested in (difficult) cytokines such as IL-10, than you can also choose to try a combination of PMA/CD3. As far as I know 11B11 is a good clone to stain with and the antibody concentration is not too crazy. PE is the right choice and the PFA/sap should not be a problem. Recomended litterature: Mouse > Assenmacher et al., Eur. J. Immunol. 1994. 24. 1097-1101 General > Prussin J. Clin. Immunol. Vol 17. No 3. 1997 195- * Maria Daly from Glaxo Wellcome in the UK replied: I can just comment on mouse splenocytes in response to antigen (transgenic mice responsive to ovalbumin peptide), or PMA+ Ionomycin stimulation. I've done a time course of cytokine production after stimulation for IFNgamma, IL-4, IL-2, IL-5, IL-10, TNFa. For my cells I find its best to stimulate the cells for a total of 4 hours in the incubator at 37 degrees C, adding monensin/brefeldin A for the last 2 hours. I find monensin is the protein transport inhibitor of choice for picking up IL-4 expression. Perhaps you could try a shorter time course. She also inluded a plot of the results she got, but this plot seems to have been lost in the transmission. *Andrew D. Wells from the University of Pennsylvania replied: I believe you are probably missing the peak of IL-4 production - IFN-g peaks later than IL-4 and IL-2, both of which are better detected 4-6 hours after restimulation. *Miguel-Angel Perales from the Memorial Sloan-Kettering Cancer Center in New York replied: I find that a 6-hour stimulation with plate-bound CD3 and CD28 in suspension (with BFA for last 2 hours) gives both IFN and IL4 responses. *Torsten Stamm from the DRFZ in Berlin replied: For intracellular detection of cytokines ex vivo or in vitro we routinely stimulate 5h with 10ng/ml PMA and 1ug/ml Ionomycin. After 3h we add Brefeldin A. We use the 11B11 PE for detection at a concentration of 3ug/ml. *Alexander Kiani from the Technische Universität Dresden in Germany replied: The question you raised is of interest for us as well. We had similar problems. Would it be possible to forward the replies you receive, or a summary thereof (in case you are not planning to post it on the list) ? *Alice Gilman-Sachs replied: In humans it is difficult to detect IL-4 in lymphocytes but easy to detect IFN-g. Could you post your answers to this query.
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