I am using gold nanoparticle in Mesenchymal stem cells. I need to measure the cellular uptake of gold nanoparticle using transmission electron microscopy. After incubating cells with nanoparticle for 24 hours how to process the cells if the sample should be given as suspension
In my opinion, you need to make a pellet cells first then you can process the sample like every biological sample
Dear Veerappan,
the most simple specimen preparation for cells in suspension has been outlined by Stefano above…
A lot of specific literature on your request "out in the wild…"
Centrifugation for cell suspension is necessary (either mixing suspension vehicle / suspended cells with suited cell fixative 1:1, let fix for some time [15-20 min @ RT will be sufficient] and spin down (e.g. 850 to 1000g for 5-10 min, preferably with centrifuge cooling) to make a pellet OR first centrifugation, producing the pellet of cells, then substituting suspension fluid with fixative solution, fixation by immersion (perhaps spinning down again @ 500-800 g for some minutes). Processing of samples as usual for TEM to get resin blocks and ultrathin sections. Only try to adjust the fixative (osmolarity, pH) for the cells you examine.
For lung macrophages & Instillation of gold particles (NB: fixation of lung tissue by cardial transfusion) cf. Gorm DANSCHER’s publication already to be found in ResearchGate @
https://www.researchgate.net/publication/40023976_Biodistribution_of_gold_nanoparticles_in_mouse_lung_following_intratracheal_instillation?ev=prf_pub ,
if you can’t find this via the URL, search in Researchgate for Gorm Danscher [https://www.researchgate.net/profile/Gorm_Danscher/] and find his publications and > Biodistribution….[article to be downloaded]>
Still more fitting to your demands perhaps:
Comenge J, Sotelo C, Romero F, Gallego O, Barnadas A, et al. (2012) Detoxifying Antitumoral Drugs via Nanoconjugation: The Case of Gold Nanoparticles and Cisplatin. PLoS ONE 7(10): e47562. doi:10.1371/journal.pone.0047562 @ http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047562 :
Personal Note: *) concerning “3000 rpm”: you certainly know that you can’t do anything with this value unless you know exactly the type of centrifuge which was used. Usually and scientifically correct “g-force” applies here (guessing it would have been around 950-1000g in this case).
Another source perhaps would be:
MIRONAVA T et al, Nanotoxicology, March 2010; 4(1): 120–137
Gold nanoparticles cellular toxicity and recovery: Effect of size, concentration and exposure time
@ http://phm.utoronto.ca/~jeffh/jnppres7.pdf ,
stating for the fixative used: [fixative modifications apply with regard to the final examination purpose]
If you have other (more detailed and / or specific) questions, please reply to the thread. Thank you!
Best wishes and good luck, Wolfgang
Article Bio-disruption of gold nanoparticles in mouse lung following...
Depending on the size of your particles you should skip OsO4 and Lead citrate staining - small ones are hard to see over the stains.
Depending on the size of your particles you should skip OsO4
Article Bio-disruption of gold nanoparticles in mouse lung following...
Abdelkader Dehbi ,it will NOT help ANYWAY to re-iterate former replies /answers, and, naturally, ist doesn‘t help the initial requester.. Veerappan Karpagam , who hasn‘t reacted to this Thread for nearly 7 years now... the Suspension presumably used in 2013 had dried out... Abdelkader Dehbi , do you have other proposals for solving the TEM-technical challenge ?? (Sent from my iPhone)
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