I have used this method before with mice T cells for detecting cytokines and added Bref.A immediately after PMA/Ionomyc. but in the literature there is a lot of differences in the time of addition. Some do it immediately and some people wait for the addition.
What is the point of waiting with adding BrefA. if you stimulate for 4-5 hrs total? Is it to rest the cells after isolation? I will stimulate human PBMCs after isolation with BD CPT tubes (density centrifugation), then stimulate them for detecting IL-17A and IL-10 in regulatory T cells and Gamma Delta T cells.
I copy and paste the paragraph from the Nature Protocol paper published in 2006 by Lamoreaux, Roederer and Koup devoted to the choice of secretion inhibitor. As you will read, it covers many aspect that drive the choice for BrefeldinA or monensin, their addiition at the start or lated during incubation, their unwanted effects.
"Brefeldin A (BFA) and monensin are the cytokine secretion
inhibitors most commonly used in ICS procedures. If peptides
are used for stimulation, either inhibitor may be added at the time
of initial stimulation because processing of peptide through the
endosome is not required. Several characteristics of each compound
should be kept in mind when choosing an inhibitor.
Monensin-treated cells are less viable when incubation periods
are more than 18 h. CD8 downregulation occurs with both
inhibitors but to a lesser extent when using BFA. BFA inhibits
CD69 surface expression but not its intracellular expression,
whereas monensin does not inhibit CD69 surface expression but
results in less expression of CD69 intracellularly. Probably the most
important characteristic of monensin, however, is that it does not
sufficiently inhibit TNF-a secretion from the cell. Therefore, BFA
must be included in the mix when examining TNF-a production.
However, because monensin is required to prevent acidification of
lysosomes and endosomes when reinternalization of CD107a
occurs during T-cell stimulation, if analysis of surface CD107a
mobilization is warranted, monensin must also be included in the
protocol. Both inhibitors can be used simultaneously without
detriment to the assay."
As you said, it might be different depending on what you do, and how you do.
I added BFA at the beginning of the incubation, for the whole 5 hours with stimulus when I used the mice T cells.
BFA is toxic to the cells to you cannot add more than 12-24(?) hours. If the cells are not very healthy, you might want to go for less hours. For the longer stimulation, I added the BFA for the last 5 hours to accumulate the cytokine inside the cells.
I have never tried Treg or gamma delta T cells for intraellular cytokine though.
+1 to Yumi. We do overnight stimualtinof mouse cells with antiben (for CD4s) and add BFA las 4-5 hours. Longer with BFA means more signal, but also more cell death. We have never had good luck with 24 hours with BFA.
We add BFA during the last 2 hours of ConA stimulated human T cells period (6 hrs max).
Add it at the beginning, then forget about it. Grab a coffee and come back 4-5h later...
Dear Alberta,
We stimulated cultured human pbmc for 6 hrs and added the bfa after 1 hr for The remaining 5 hrs. We had good results with that procedure. We stimulated with either pma/ionomycine or anti cd3/cd28.
Good luck with your experiments,
Jeroen
After stim with PMA/Ionomycin for 6 hours we add the BFA during the last 2 hours of incubation .
It also depends on the cytokine which you want measure later by FACS. For some cytokines only BrefA stimulation is enough without PMA/Ionomycin. But for IL-17, PMA/Ionomycin stimulation is necessary. I usually use BrefA along with PMA/Ionomycin for 4hrs for detection of IL-17A.
It really depends what you want to measure. During BFA incubation you block secretion of produced cytokines hence they accumulate in the cell. If you wait with addition of BFA after PMA/Iono you will lose the cytokines secreted immediately either due to the stimulus from PMA/Iono or any other. Which schedule is better? You have to find out for you cytokine of interest.
When looking at T cell production of IFN-y, IL-2, TNF and IL-17, I usually stimulate with PMA/Ion for 5 hrs. I add BFA after the first hour of incubation. If you are looking at cytokines post 3 day TCR stimulation, I would add BFA in the last 4 hours of that incubation.
As i understand BrefeldinA is a vesicular transport inhibitor. Usually used to inhibit export of vescicular cytokines such as IL-1a. However BFA as been used to understand release of IL17. But this cytokine is not stored in vesicle. Usually IL17 release is detrmined at 48-72 hrs post PMA/ionomycin stimulation. BFA is added at last 4-5 hrs of measurement.
For stimulation of PBMC isolated from EDTA blood I use a 4 hour PMA/Ionomycin stimulation, with Brefeldin A added at the start of the culture.
then check for combinations the following cytokines: IFN-gamma, IL-17A, Il-4, IL-2, and IL-21 or IL-22.
I have also cultured for 5 hours with Brefeldin A from the start, and the results were the same.
I think an intial culture period without Brefeldin A is to let the cells equlibrate/recover from isolation. However, it could allow for modulation of surface antigens. By adding brefeldin from the start you will prevent upregulation/down-regualtion of PMA/Ionomycin responsive surface antigens.
I see some IL-17A after 4 hours, typically being produced by CD4+ memory cells. I'm afraid I don't have any experience with IL-10.
I use the eBioscience Foxp3 kit to fix and perm the surface stained PBMC, and it works well, and allows you to combine Foxp3 with cytokines.
It depends on what you're looking for. If your target of interest is downstream in the activation process and relies on the full and complete synthesis of another marker first you will need to wait an approriate time before adding Brefeldin A. If its produced immediately on activation, then you'll need to add Brefeldin A immediately. Its been a while since I've done it by I found higher levels of IL-10 when I used Monensin instead of Brefeldin A.
Hi, I have been using ConA for stimulation of rat splenocytes. Initially I incubated with brefeldin a few hours after ConA stimulation, but could not detect the cytokines I was interested in (IL4 and IFNgamma), no matter how long I incubated with brefeldin (ie 2 hours, 4hours, 8hours, 10hours, 12 hours after initiation of culture). So, my next step was to actually follow the kinetics of conA mediated stimulation and found out that the best way is to stimulate for three-four days and add brefeldin 12 hours prior to analysis. My advice therefore would be that if you are having trouble detecting the cytokines of your interest, it is worth optimizing the stimulation conditions and allow your cells to be stimulated enough to produce significant levels of cytokines. Good luck!
dear Alberta,
I stimulated cultured human PBMC or gammadelta cell lines with PhAg, PMA/Iono, or aCD3/aCD28 (for total 6 hours and also 18 hours) and after one hour from stimulation I added BFA. I obtained good results for inflammatory cytokines (TNFa/IFNg), and also for IL10. I didn't analyze IL17A.
Eleonora were you able to detect IL-10 in gamma delta T cells? as most people are pessimistic about the abiliity to detect it by flow and by the PMA/İonomyc.BrefA method.
I tried in human pbmc and intrahepatic gammadelta T cells only for unstimulated cells. I observed a frequency of Vd2+/IL10+ about 1-2%.
I copy and paste the paragraph from the Nature Protocol paper published in 2006 by Lamoreaux, Roederer and Koup devoted to the choice of secretion inhibitor. As you will read, it covers many aspect that drive the choice for BrefeldinA or monensin, their addiition at the start or lated during incubation, their unwanted effects.
"Brefeldin A (BFA) and monensin are the cytokine secretion
inhibitors most commonly used in ICS procedures. If peptides
are used for stimulation, either inhibitor may be added at the time
of initial stimulation because processing of peptide through the
endosome is not required. Several characteristics of each compound
should be kept in mind when choosing an inhibitor.
Monensin-treated cells are less viable when incubation periods
are more than 18 h. CD8 downregulation occurs with both
inhibitors but to a lesser extent when using BFA. BFA inhibits
CD69 surface expression but not its intracellular expression,
whereas monensin does not inhibit CD69 surface expression but
results in less expression of CD69 intracellularly. Probably the most
important characteristic of monensin, however, is that it does not
sufficiently inhibit TNF-a secretion from the cell. Therefore, BFA
must be included in the mix when examining TNF-a production.
However, because monensin is required to prevent acidification of
lysosomes and endosomes when reinternalization of CD107a
occurs during T-cell stimulation, if analysis of surface CD107a
mobilization is warranted, monensin must also be included in the
protocol. Both inhibitors can be used simultaneously without
detriment to the assay."
In fact the incubation time range of BFA in the culture mainly depends on the used concentration for example 5ug/ml to 10ug/ml the incubation time 6- 10 hours is normal even 12 to 16 hours there is no problem, but at this concentration you should avoid incubation more than 18 hours, because it well be toxic for your cells. While more concentration requires less incubation time. in general the recommended concentration is from 5 to 10ug/ml which endows a wide range of incubation time.
Hi,you can add BFA 10 microgr/ml also after 1 hour from stimulation and you may try 6h and 18h of colture to detect cytokines by flow cytometry in humans. Bye!
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