Hi everyone!
I've been trying to optimise a CD11b antibody, using THP-1 cells. In the latest experiments, I've spiked whole blood from healthy donors with differentiated THP-1 cells, but I get no signal from them, while other WBC show some degree of expression of CD11b.
What can be wrong here? I'm assuming the differentiation protocol hasn't been working properly. I've used PMA (200 nM) as an inducing agent, for 3 days + 5 day resting period with fresh medium.
Any thoughts on this issue will be much appreciated!
Thank you in advance for your time!
João Maurício
I feel the concentration of PMA is too high. I have been using 50nM for about 24 hours without a change in medium.
I feel the concentration of PMA is too high. I have been using 50nM for about 24 hours without a change in medium.
I am using 100 nM PMA for 72 hrs for differentiation without changing media. After PMA differentiation you can try to stain for CD11 with fluorescence antibody and analysed on flow cytometre .
I think you are using maximum concentration of PMA but for longer period. You can use either 200 nM for 48 hrs with 10% FBS or 50 nM for 48 hrs without serum this will increase the differentation capacity again depending upon your expt requirement. Thereafter after washing with PBS you can check CD11b antibody. Use 2-betamercaptoethanol minus media at time of differentiation. Further CD11b staining protocol should work optimal at 40C for 30 min in HBSS Ca2+ BSA buffer. Hope these strategies will work.
Joao,
My group has carried out much work on the differentiation of THP-1 cells. In general, our starting point was 50ng/ml because of the standard use for T cell activation. From this starting point we had a look at varying doses/times. The main point of consideration is that PMA is an analogue of DAG that can be involved in signal transduction resulting in the activation of PKC and a potential to activate apoptotic pathways also. With this in mind, we had a good look at the dose/time most appropriate for differentiation without inducing cell death. Your concentration of 200nM (which equates to approx 123ng/ml, is rather high and can induce cell death in THP-1s). Putting together with time of differentiation, we found an appropriate differentiation towards pro-inflammatory macrophages by using 25ng/ml (approx 40nM) for 3-4 days, but, as you are aware, because PMA is a stimulatory factor, there is a need to wash the PMA out. This can be appropriately achieved by 1-2 days washout in fresh culture medium. This washout period returns the now differentiated cells to a resting background level that will allow for measurment of activatory responses initiated in these differentiated macrophage-like cells. If you need any further info, contact me by e-mail. How is Newcastle? Regards, Andy.
I used 100 nM PMA for 48 h. Check for morphological changes under microscope then stain with Ab
protocol for differentiation:
https://www.ncbi.nlm.nih.gov/m/pubmed/27784678/?i=1&from=anberitha%20matthews
There might be some discrepancy on what people suggest to use as a concentration. This is most likely due to the quality of the stocks as well as the integrity of the compound. PMA is acylated at two sites and are prone to hydrolysis. I get great results with 1uM PMA after 3days. I have not looked at a 48hr time point, so I can't comment on that.
1. THP1 were added to 96-well plates: 140uL volume (75,000 cells)
2. Cells were treated with PMA (10uL volume) final concentration 1uM.
3. Following a 3-day incubation period, 50uL of a staining solution (CD11b to achieve a final concentration of 1:100 and DAPI 1:1000) were added to each well for 45 min.
4. Cells were then fixed with 50uL of 20% PFA = 4% final concentration for 15min at RT
If you need more details feel free to email for full protocol.
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