I am working on hPL, after hPL treatment cell morphology is changed, cells look smaller and little stressed. There is fine deposition of granules on and around cells in hPL treated wells. This may be due to gel formation which I think can be solved by adding heparin. Is the change in cell morphology is because of the gel deposition?
Do you want to prove that hPL has factors which caused morphology change in target cells? You would have to purify the hPL and test each purifed fraction on target cell morphology. Is it necessary to use hPL? What happens if you co-culture intact platelets with the target cells? Is there any change in target cell morphology? Hope this helps!
As the idea is to use platelet lysate the co-culture may not be a solution.
Fractionating to find the factors responsible will defeat the purpose as it would add to cost.
I see your point. Co-culture may help you decide when platelets are in perfect health--and lysate can be made at that point.
Do you expect improved wound healing by adding hPL to target cells? Improved wound healing may not take place if cell morphology is bad. So, best to focus on media conditions of target cells, health of platelets before they are lysed. Storage media (if any), being used. Density of cells seeded, frequency of media change..Perhaps one or more of these conditions are critical to get perfect lysate!
We found that stored RBC's were more viable when they were rocked at 4degC rather than kept stationary. NO release was better during rocking! Hope this helps!
All the factors mentioned above do affect the quality of hPL. So most of the things are kept constant across the expt.s. When hPL is added to healthy cells, they shrink ie their vol looks smaller. I wounder is this the hPL phenomena or something else?
From your observation from RBC would you suggest that stored rocking at 4deg C hPL may stay better? worth experimenting.
Reg adding hPL to healthy cells: It could be an osmotic change. hPL contains many charged molecues (proteins). What about salt? Has conductivity been done? Is it possible to dilute or dialyze hPL and loose this unwanted effect on target cell? Will hPL's activity also get lost upon dilutin /dialysis??Does equivalent concentration of BSA have this unwanted effect?
Reg sotrage on hPL prior to making lysate: RBCs prefer mild rocking/shaking because this activates NOS and NO is released..RBC thrive in presence of NO. Is there similar effect of NO or any small molecule on intact hPL? cells: I dont know the biology of these cells. But, its worth reading and trying different methods of storing the hPL prior to lysing. Sitting at 4deg C may not be ideal !
Hope this helps!
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