Dear Shaima Sirajudeen,

I wonder about your previous reading some (hand)book(s) or at least one of the hundreds of protocols for TEM-preparation/processing of cultured cells, therefore also for HeLa cells. 

But it might be easier to ask for a solution or "a good protocol" via RG than to perform a thorough literature search on scientific work already done....

Even as a newcomer you should have read specific literature about what and how you will be processing and examining:

Classically cultured cells (dish), or culture on grid, classical TEM (morphology) only or TEM-IEM, or proving physiological cellular processes, etc.,etc.

And, honestly, then at least then you should have seen some TEM recipes/ processing schedules matching your specific needs. 

So only as a rough orientation:

Pellets (which requires i) careful [mechanical?] collection of cells, and ii) careful centrifugation prior to or during fixation, and iii) careful - and thus tedious - processing by further processing of the pellet (if not using e.g. the agar-embedding-method for cell [culture] pellets).

For classical TEM of dish-cell culture:

usually one would either suck off culture medium and overlay carefully and thoroughly a suited fixative (depending on the intended further experimental needs) or dispense fixative with double concentration (1:1) to the culture medium...  concentrations: 0.5 - 1% FA and / or FA/GA (0.5-1%)  in a suited buffer, @ correct tonicity and pH and either @ RT or at 4°C (choice depending on exptl. design), duration of initial fixation: 5 to 10 min. Appropriate washings and further processing (+ or - OsO4 )as usual for TEM-processing into (?epoxide, acryls, etc.) resin.

Only as one recent reference (as an example for localizing nanoparticles in cultured cells):

"Preparation of cells for assessing ultrastructural localization of nanoparticles with transmission electron microscopy"

Amanda M Schrand , John J Schlager , Liming Dai & Saber M Hussain

Nature Protocols 5, 744–757 (1 April 2010) | doi:10.1038/nprot.2010.2,

 to be found at (restricted access to subscribers or PPV=pay per view):

http://www.nature.com/nprot/journal/v5/n4/full/nprot.2010.2.html

Best wishes and good luck.

Dear Dr. Wolfgang

Thanks for taking time to answer my question. I have done the literature work and am well aware of the steps. But I encountered sufficient loss of pellet during the dehydration and resin infiltration steps. The reagents are costly and the process is time consuming, so I couldn't keep on trying with different variables. That's why I put up the question on RG.

I have tried a few protocols, but the cell pellets kept on decreasing in volume as I advance through the dehydration and resin infiltration steps until at last I have little or no sample to do the TEM. That is why I'm looking for a protocol that can prevent this problem. When I did the centrifugation for the cell pellets at 1,000 rpm for 5 minutes, the cells did not seem to settle well. When I increased the centrifugation time, the cell pellet reduced in size and I couldn't do the TEM.

Shaima,

Have you tried agarose embedding of your pellet after primary fixation?  For light pellets this is the way to go, usually 1-3% agarose (heated to solubilize).  Also with some cells I find they don't pellet well after fixation, in this case I fix in suspension for 10 min, then spin down to a visible pellet (1300 G), replace with fresh fixative and continue to fix the pellet.  I usually find I can gradually increase the speed of the centrifuge after secondary fix, and definitely when I get to spinning down in resin.  Live cells are usually pelleted at about 1300-1500 G. Good Luck

Good sugestion Michael.

We pellet GA-fixed cells 1 minute 1100 x g. Discard the supernatant and add an equal volume of warm (but not too hot, ~55°C) 4% low melting agarose in phosphate buffer. Mix and spin down for a few seconds. I have the centrifuge at 37°C during this step. Tubes are placed in melting ice.

Then I saw away the upper part of the tube and take out the agarose w/ cells, slice it ~0,5 mm thick w/ a surgical knive. The slices are submergded in cacodylate buffer or phoshate buffer for 30 minutes and then proceed to OsO4 fixation.

Hi Duco

Thank you for the suggestion and taking time to answer my question.

Thank you very much Michael.

Good luck Shaima. If the suggestions are valuable you may also up vote them (the little green arrowhead just below the answer), any realy stupid, or abusive remark is easily down voted. It is also very much appreciated to give a little update afterward. Other RG-users can learn from that too.

Dear Shaima:

thank you for your friendly and informative reply. Thanks also for not taking my reply as offending (since my answer by no means was intended to be such!!).

Now that you revealed the problems you were faced with the experts will be able to help for sure more in detail. Michael and Duco (thank you for chiming in) have given some valuable and practical hints, especially on the -embedding (which in fact is the same as the agar-embedding I mentioned in my post) method.

One question left for me: your centrifuging cells is done on a >swing out-> or centrifuge? Because there could be another parameter waiting to be addressed (loss of cell material).

 Just as one example of the agar-agarose embedding method I enclose here for your convenience:

"AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix", by Harold M.J. Kerstens, et al, 

J Histochem Cytochem 2000 48: 709; DOI: 10.1177/002215540004800515 ,

kindly provided by JHC-SAGE Publications =   http://www.sagepublications.com and open access  @ http://jhc.sagepub.com/content/48/5/709

 There is a similar article published in  ULTRASTRUCTURAL PATHOLOGY,  Volume 30 Number 5  Sep-Oct 2006  p. 379  - 385 ;     URL of abstract:               http://www.tandfonline.com/doi/abs/10.1080/01913120600854269  

by  Soheir S. Mansy, Mahmoud A. Abbas, Hoda A. Yehia, Samia M. AbdelRazik, Lobna Y. Ghanem, Tarek M. Amin

"Value of the Innovated Technique Agarose Cell Block in Improving the Sensitivity of Urine Cytology in Cases of Bladder Carcinoma"

Abstract: Proper handling and processing of urine sample can greatly improve diagnostic sensitivity. This work investigates the value of agarose cell block technique in processing urine samples simultaneously for light and electron microscopic examination, with the prospect to enhance the quality of diagnosis. The material of this study consisted of 45 voided urine samples, processed for the performance of Papanicolaou-stained urine smears, agarose cell blocks paraffin sections stained with hematoxylin & eosin, and electron microscopy-contrasted ultrathin sections. The studied technique increases the sensitivity of urine cytology and opens a new prospect for cytomorphological study.

Keywords: agarose cell block technique, electron microscopy, urine cytology

but unfortunately that article only available either as subscriber or by PPV (pay-per view only). Wishing you all the best with trying the -embedding technique, good luck and perhaps you come back to your thread when you can tell about hopefully positive results.  

Best regards, Wolfgang  (you don't need to use the "Dr.")

Dear Duco

Thankyou very much for the advice, I really appreciate it. I will update as soon as I finish my next experiment. 

Shaima

Hi Wolfgang

Thank you for taking the time to reply me again in a more supportive fashion.

 The centrifuge I'm using is one with a stationary rotor. And thank you very much again for the useful links you provided.

Good morning/Good day,

Dear Shaima, your'e welcome!

I see: stationary rotor (high speed rotor for Eppendorf or Eppendorf-like tubes? cooled centrifuge? brake function? But for sure  you did centrifugation for about 5-7 min at 1000-1100g?). Your 'pellet' does form laterally on the E-tube wall....not at the bottom, right?

The 'evil' parameters of centrifugation: smaller / older centrifuges (especially if they have been in use for a longer time without necessary service) might be a bit out of center or run with a bit / slightly worn-out bearings which result in uneven-imperfect if not impossible/unfeasable sedimentation/pelleting of cells from culture.

So - naturally - when it comes to changing solutions/washings you will  loose a lot of cells (and you won't see that clearly unless the cell pellet is fixed and osmicated...   therefore a good option will be the agarose embedding technique which Michael and Duco mentioned practically.

All best wishes and good luck

Wolfgang