The background information of our study is that I need to compare the proliferation of peripheral blood mononuclear cell (PBMC’s) in two groups of samples, namely, with antigen (PHA – 50µg) and without antigen. Again each of these two experimental groups had two different concentrations of initial cells (10,000 and 100,000, respectively). The antigen challenge was given for 48 hrs. BrdU was incorporated in the last 2 hrs of challenge. But there was hardly any difference in the absorbance values with antigen and without antigen. I am unable to infer anything from the obtained results. Please suggest, if I need to increase the concentration of the antigen or time duration of the challenge? Kindly help me to get a solution in experimental design a well as analysis of the data, thereof.
I am attaching the image of plate and the data obtained.
Experimental Design
1. Peripheral Blood Mononuclear Cells (PBMC’s) were isolated from human blood using Hisep 1077 by density gradient centrifugation method.
2. Viable Lymphocytes were counted using Haemocytometer.
3. Cells were placed in a concentration of 10000 and 100000 with and without antigen.
4. The antigen used was Phytohaemoglutinin (PHA) at a concentration of 50µg.
5. PLATING FORMAT :
Wells with Antigen
Wells: A2, A3 and A4 – Blank (150µl media + 50 µl PHA)
Wells: B2, B3, B4, C2, C3, C4, D2, D3 and D4 – 10000 cells/well in 150µl media + 50µl PHA
Wells: E2, E3, E4, F2, F3, F4, G2, G3 and G4 - 100000 cells/well in 150µl media + 50µl PHA
Wells without Antigen
Wells: A7, A8 and A9 – Blank (200µl media)
Wells: B7, B8, B9, C7, C8, C9, D7, D8 and D9 – 10000 cells/well in 200µl media
Wells: E7, E8, E9, F7, F8, F9, G7, G8 and G9 - 100000 cells/well in 200µl media
6. After 48 hrs. of antigen challenge the plate was centrifuged at 1500 rpm for 20 mins. and 100µl of media was removed following the addition of 10µl of 10x BrdU labeling reagent to each well, mixing and incubating for 2 hrs.
7. After the completion of incubation the plate was again centrifuged at 1800 rpm for 20 mins. and the media was discarded carefully.
8. Plate was dried for 15-20 mins. using a blower, till all the media was dried up.
9. 200µl/well of fixing/denaturing solution was added and the plate was incubated at 25ºC for 30 mins.
10. The solution was discarded with wrist flick and the plate was tapped on an absorbant paper.
11. 50µl/well primary antibody was added to each well and the plate was incubated for 1 hr. at 25ºC.
12. Removed primary antibody and 5 washings as per the protocol.
13. 50µl/well secondary antibody was added to the wells of columns 3, 4, 8 and 9 (wells of column 2 and 7 were added with 50µl/well triple distilled water).
14. Plate incubated for 1 hr. at 25ºc.
15. Removed primary antibody and 5 washings as per the protocol.
16. Last wash with 300µl/well with 1X PBS. Removed with wrist flick and tapped onto absorbent paper.
17. 50µl/well substrate reagent was added to the wells of columns 2, 4, 7 and 9 (wells of column 3 and 8 were added with 50µl/well triple distilled water).
18. Plate was incubated for 15 mins. at 25°C
19. After completion of incubation 50µl/well stop solution was added to each well.
20. The absorbance was measured at 450/540 nm, 450/550 nm and 450/595 nm using an automated ELISA reader.
I am attaching the images of ELISA plate and the data carrying the absorbance values at the required wavelengths.
Points of discussion:
As according to the manual it is a comparative analysis checking the proliferation of the cells with and without antigen but there is hardly any difference in the values even after 48 hrs. of antigen challenge.
Do I need to make any changes in the protocol to get some appropriate difference?
Hello!
I have been using a similar kit for some years now, also for human PBMCs. I think mine is not exactly the same kit as yours, since there are some minor differences, but, besides the volumes, the protocol is the same.
From my experience, your problem is not the PHA stimulation, but the incubation with BrdU. I performed an experiment when setting up the protocol to test different BrdU incubation times and the optimal incubation time with BrdU was overnight (around 14 hours) for 100000 PBMCs. So what I am doing is to add the BrdU on the last night of the PHA stimulation (I also stimulate for 48 hours, and even for 72 hours for some experiments) and to perform the rest of the protocol (drying the plate, fixation/denaturalization, etc.) in the next morning. I am easily getting absorbances of around 0.9-1.2 (450 nm - 620nm), much more higher than yours
I think you can try some experiment allowing more time with the BrdU (you can maybe try several different timepoints) and keep the rest of the protocol unchanged, so you can see if longer incubations improve your results.
Hope it helped!
Regards.
Nuria
Thanks for your prompt response. Looking for further suggestions.
Sorry I did not have chance to go through your whole experiment. BrdU gets incorporated only when the DNA in the cell is replicating, so the pulse chase time should be determined based on the expected dynamics of the cell cycle. If you added the BrdU after the cell has passed S phase, the incorporation will not take place until the cells undergo the next round of replication (DNA synthesis).
Thank you all for the replies.
We have set the xpriment for 18 hours (both control and treatment groups). However, the major problem encountered is that I am getting almost the similar absorbance values for unchallenged cells as for challenged ones (attached).
In other words, I have even tried using 18 hour incubation of BrdU but there is only marginal difference between the two groups. So, my specific query is whether the control group should also show absorbance or not? Please reply.
Your protocol suffers from different problems:
The short incubation time which may be one of the most important problems has been cured. We use proliferation tests in a clinical setting and allow routinely 16-20h of BrdU incorporation.
The next important step is to remove ALL unbound BrdU from your cells as most antibodies do not differentiate between BrdU incorporated in DNA or being unboing in the well. Wash your cells several times with PBS with 0.05% of TWEEN. You have to centrifuge your plates between every washing step to avoid cell loss. Do not tap the plate on paper towels etc. as this will cause cell loss! We do not use a kit, so our last step is to fix the cells with ice-cold ethanol. Again, the plate is centrifuged to have all cells at the bottom of the well. This may be important for proper BrdU detection by the antibody.
After drying the plate, denaturation is an important step. You may test either HCL (1-2N) or microwave, both work well in our hands. If you do not see any colour development adding your chromogen do not stop the reaction but wash your plate and start again from the denaturation step.
It is also highly recommended to add a control without BrdU to get the background values of your antibody. Subtraction of this background from the whole plate values is important in the plate analysis.
There are several forms of PHA which have varying capabilities to stimulate lymphocytes. Overdoses of PHA will kill the cells. Using PHA-L which is the most potent stimulant the concentration should not exceed 5µg/ml. Optimal doses of PHA-P or PHA-M have to be estimated as they are crude preparations with varying stimulatory capacities. Anyway, 50µg/ml seems to me as a very high dose.
I hope that these suggestions may help to solve your problems.
regards
Gernot
Hi. I have a question. Why do you read at 595 if the signal is at 450nm.
I should include the values at 595 nm in my calculations?
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