6 replies to this topic

[iamnewhere]

hi there.. i'm new here and im also new in cell culture..

i have problem in coating 6well plate with poly-D-lysin(PDL)from Sigma...
I usually coat 50ug/ml of PDL in 1 ml per well.. then I incubate in hood (room temp) for 24 hr.
after 24 hr, i aspirate the PDL solution n rinse with 1ml PBS/well. then I put it aside n prepare for cell seeding.
the next day, I found that some of the cell is detach and only half of them were attached.

1) is there probably due to contamination of the plate?
2) I directly put the cell into the well without allowing the plate to dry. is it because of that?
3) I rinse the PDL coated plate with PBS instead of distilled water. is it ok? does PBS give affect to the Poly-D-Lysine?
4) In order to avoid contamination, can I filter the PDL solution before coat the plate?

I hope to get immediate feedback from you all that have experience or expert in this topic..
thanks a lot for your concern and kindness..

[smr86]

Hi,

In our lab, we apply poly-D to the plates for 30min, then remove and wash plates 3 times with dH2O, leave the plates to airdry in the hood for approx 1h, then store at -20C until needed.  I don't know whether any of the points you have raised could be causing a lack of adherence.  Sigma don't suggest to use PBS in the protocol so there is probably a good reason for this.  Also, although I mentioned that I leave my plates air-dry, I should also mention that I thoroughly remove each water wash so the plates are practically dry when I leave them to airdry.  Following this protocol, we have never had any problems with cells detaching.

[knuf]

How we've done it is the day before, filter sterilize 100 ug/ml PDL diluted in water and apply them to the plate and put them in the incubator. Then before plating the cells, remove the PDL, rinse 3 times in sterile PBS and apply the cells. We do this slightly differently if we are doing glass coverslips, though.

[iamnewhere]

smr86, on 27 April 2011 - 03:40 AM, said:

Hi,

In our lab, we apply poly-D to the plates for 30min, then remove and wash plates 3 times with dH2O, leave the plates to airdry in the hood for approx 1h, then store at -20C until needed.  I don't know whether any of the points you have raised could be causing a lack of adherence.  Sigma don't suggest to use PBS in the protocol so there is probably a good reason for this.  Also, although I mentioned that I leave my plates air-dry, I should also mention that I thoroughly remove each water wash so the plates are practically dry when I leave them to airdry.  Following this protocol, we have never had any problems with cells detaching.

hi smr86.. thanks for your concern.. ...
you have mentioned that you apply the poly-D to the plate for only 30 mnt.. I think it is better compare to 24 hr incubation in terms of time saving.. I think I should try that..  
is it ok if you wash your plates 3 times with dh2o? does it detach?
when you leave them to airdry, did you take off the plate cover?

[iamnewhere]

kfunk106, on 27 April 2011 - 11:23 AM, said:

How we've done it is the day before, filter sterilize 100 ug/ml PDL diluted in water and apply them to the plate and put them in the incubator. Then before plating the cells, remove the PDL, rinse 3 times in sterile PBS and apply the cells. We do this slightly differently if we are doing glass coverslips, though.

hi kfunk106.. .. thanks for concern..

-i used 50ug/ml of PDL... does different concentration give different effect to the attachable of the cell?? is it true if we use higher concentration, the cell will more attachable compare to lower cncentration... but if Im not mistaken, if the concntration of PDL is too high, it might be toxic to the cell..

did you use 0.2 or 0.45 filter? does the protein filtered too??

[knuf]

iamnewhere, on 01 May 2011 - 07:54 PM, said:

-i used 50ug/ml of PDL... does different concentration give different effect to the attachable of the cell?? is it true if we use higher concentration, the cell will more attachable compare to lower cncentration... but if Im not mistaken, if the concntration of PDL is too high, it might be toxic to the cell..

did you use 0.2 or 0.45 filter? does the protein filtered too??

That may be true. We were using this concentration for neuronal cultures, so there may be differences in toxicity or concentration necessary depending on the cell type used...I really can't speak to that. As for the filter, we used a 0.22 um filter. I wouldn't think that the pdl would be large enough to get stuck on the filter, but I've never tested it either.

[smr86]

iamnewhere, on 01 May 2011 - 07:45 PM, said:

smr86, on 27 April 2011 - 03:40 AM, said:

Hi,

In our lab, we apply poly-D to the plates for 30min, then remove and wash plates 3 times with dH2O, leave the plates to airdry in the hood for approx 1h, then store at -20C until needed.  I don't know whether any of the points you have raised could be causing a lack of adherence.  Sigma don't suggest to use PBS in the protocol so there is probably a good reason for this.  Also, although I mentioned that I leave my plates air-dry, I should also mention that I thoroughly remove each water wash so the plates are practically dry when I leave them to airdry.  Following this protocol, we have never had any problems with cells detaching.

hi smr86.. thanks for your concern.. ...
you have mentioned that you apply the poly-D to the plate for only 30 mnt.. I think it is better compare to 24 hr incubation in terms of time saving.. I think I should try that..  
is it ok if you wash your plates 3 times with dh2o? does it detach?
when you leave them to airdry, did you take off the plate cover?

Hi there,
Well we've never had any problem with adherence after 30min.  In fact, the product information on Sigma's website recommend only 5min, I suspect though that lab members may not have been happy with this when they first started to use it and so increased the time to 30min.  

With regard to water, all I do is distribute H20 into the wells from a 25ml pipette (not paying any attention to volumes-it is just a wash after all!!).  As I normally do 6 plates at a time, I put on the wash to all plates, wait a minute then remove the wash again using a glass pasteur pipette attached to vacuum pump.  You do need to be careful with this step I find so as not to scratch the plate surface when removing the wash.  I just tilt the plate at a slight angle and place the tip of the pipette at the base of each well and remove the water like that.  For air-drying, I leave the plates in the hood with the lid slightly ajar, to avoid any accidents if others are using the hood after me.

With regard to poly-D concentration we use it at 0.1mg./ml for neuronal cultures.

Good Luck!

smr86