failed Plaque Assay! what do I do wrong? - my plaque assays fail one after another!!! (Jun/01/2008 )
my plaque assays fail one after another, it is just ridiculous,....however, it's a long post and I guess no one will read it to reply
basically I do the plaque assay as follows:
1- I make a 80-90% confluent 6-well plate of HeLa cells,growing in DMEM+10%FBS+1%antibiotics
2- I make a serial dilutions of my virus in PBS (I have a paramyxovirus and basically what I do is that I take 10ul of the pure filtered virus and top up to 1ml, then take 100 ul of this sample and top up to 1ml to make my second sample, and so on...I make 5 samples)
3- I aspirate off the medium
4- add 100 ul of each sample to each well and leave the last well as my control.....then add 200 ul PBS to each well to make sure the virus can spread all over the wells
5- I put in incubator for 1 hour, 37 degrees, 5% CO2
6- in the meantime I make 25 ml DMEM+2%FBS+1%antibiotic and put in 50 degrees celcius water bath in a 50ml Falcon tube
7- at the same time prepare a 2% agaros gel, autoclave
8- I add 25 ml of this agarose to the 25 ml of my DMEM in water bath to make a final 1% gel
9- I aspirate off the inoculum virus after 1 hour and wash with PBS once
10- I add 2ml of the mixture of gel when I feel the temperature is ok by touching the falcon tube
11- let to cool down for 5 minutes and then put in incubator
12- leave for 5 days (however I check the cells under microscope everyday and on the 4th day I see that many cells have died under the gel. a friend of mine also confirmed this, he does plaque assay routinely,and told me once that I can not see any plaque because my cells have all died )
13- on the 5th day I add 1 ml of 3.7% Formaldehyde on the gel, leave for 4 hours
14- aspirate off formaldehyde, lift the gel very carefully, not to disturb the cell monolayer
15- I add 1ml 0.05 % Neutral Red, and then shake the plate briefly to make sure Neutral Red spreads in the well (but when I do this many cells lift up, does it mean that the cells are not fixed yet?, or if the cells have died then they never get fixed even with Formaldehyde?)
16- I see no plaque after turning the plate upside down to let neutral red dry
What do I do wrong?
Do you have any idea of the titre of virus you are using? Borrow some virus from a colleague and try a plaque assay with that. Maybe you dont have enough infectious virus in 10 ul of the original inoculum. The assay procedure itself sounds ok to me, not sure why your cells are lifting after fixation though. Is it necessary to have FCS in the overlay medium during the plaque assay? This can inhibit plawue formation for some viruses....double check with published protocols. Also, do not move the plates every day. If there is any liquid trapped under the overlay.....as is often the case, moving the plates will prevent discrete plaque formation. Also i just noticed you use DMEM in the overlay, we use 2XMEM, so when it is diluted with the agarose it is at 1 X concentration.....maybe your cells are not receiving enough nutrients??
thanks avalon
I'm going to do this procedure again with a friend of mine, he knows how to do it. he also suggests that I've got no virus at all, but i checked the pure virus on SDS-PAGE and I have good virus bands.........so the virus is there, but don't know if it's active or not
is the pH of Neutral Red important?
I didn't get result again, I used 0.5% Neutral Red this time. still can't see the plaques.
I checked the wells under microscope, the control cells were still alive, and the infected ones were dead mostly. so the plaque must be there, but I think my method of visualization is wrong.
Are the cells mostly dying in the centre of the wells?? 300ul doesn't sound like enough to cover cells in a 6 well plate properly- maybe your cells are drying out??
no they die in everywhere, I'm going to use Crystal Violet this time, not Neutral Red. I have a plate in its 7th day, I'm going to add Crystal violet today.
no they die in everywhere, I'm going to use Crystal Violet this time, not Neutral Red. I have a plate in its 7th day, I'm going to add Crystal violet today.
Fix monolayers with 10 % Neutral Buffered formalin ( ie add this on top of the agar) for one hour in a fume hood. Remove the NBF, flick the agar off the plates and stain with 1% crystal violet in ethanol for 2 min (just enough to cover the bottom of each well). "dunk the plates into 3 tubs of water to remove the crystal dye and u should be able to see plaques if they are there. Also, they should not be dying everywhere in a plaque assay or you are using too much virus!! The idea is to use a dilution where you can count clearly isolated plaques....not to kill everything off
ok, thank you, I have an HT29 plate which will be 90% confluent by tomorrow. I will start all over again.
the last experiment with crystal violet didn't give plaques. but I managed to stick the cells to the bottom of the plate by fromalin successfully.
It would be better if you dilute the virus in serum free DMEM instead of PBS too
I thought DMEM doesn't let virus attach to host cell surface/receptors, and that is why we wash the cells before adding virus+PBS.