By the way, I want to know if the nuclear protein (LAM A/C) express when the cell dead.

Hi, I suggest Live/dead viability assay kit.

LIVE/DEAD® Viability/Cytotoxicity Kit—Membrane-permeant calcein AM is cleaved by esterases in live cells to yield cytoplasmic green fluorescence, and membrane-impermeant ethidium homodimer-1 labels nucleic acids of membrane-compromised cells with red fluorescence.

Thank Asli and Subhadeep, i want to know which concentration is better for coatch dish with PLL? And my purpose was determine if the nuclear protein (LAM A/C) express when the cell dead. And are there some suggestions about kill cells? Thanks again.

Propidium Iodide (PI) will label the nuclei of dead cells. Its similar to ethidium bromide but PI is routinely used to label dead cells for FACS sorting etc.. and works quite well for visualisation of dead cells. You can use it in combination with Calcein AM which will label live cells instead of buying in a live/dead staining kit (PI and Calcein is essentially what the live/dead kit is anyway). PI is commonly used so I'm sure someone around your institute will have some.

I would suggest labelling your cells in suspension (as you would do for FACS) and do a cell spread on a slide. There are lots of ways of killing cells..camptothecin, staurosporine..if you want them to go through apoptosis and die.

I'd suggest you using Alexa Fluor® 350 Carboxylic Acid, Succinimidyl Ester by Invitrogen. It randomly stains proteins and due to the missing membran integrety dead cells appear to be brighter then living cells. To me this approach is more suitable if you are restricted to a certain antibody. Alexa dyes have a discrete spectrum in contrast to PI, so you can combine AF350 with virtually any antibody.

See this Link:

http://products.invitrogen.com/ivgn/product/A10168?ICID==%3Dsearch-product

There are several assays to monitor dead cells. I would suggest to use Annexin V-Fitc/PI detection kit is very easy to use for both attached and in suspension conditions.

ANNEXIN V-Fitc Apoptosis detection kit from Sigma Cat# A2214

As you described your experiment most likely the detached cells disintegrated and turned into a cell debris (no pellet after centrifugation); all epitopes are lost, and they cannot be stained. Look at them in a hemocytometer to check if it is true. I suggest, use earlier time point and less H2O2 to stain dying but intact cells while they are still attached. Good Luck.

If you want to stain the nucleus of dead cells,you can centrifuge the cell suspension and prepare either an air dried smear or wet smear.If you are air drying the smear you can try any of the Romanowsky stains like geimsa,leishman etc.If you prefer wet preparation you can try with hematoxylene stain, which require fixation in 95% alcohol or its substitute.You need not go for immunostaining

Thanks for all answers. I want to know if dead cells express Lamin A/C. It was confirmed live cells express lamin A/C.

Dear Chao Chi, just to comment your last post: perhaps it would be useful to be informed about the cell type you are working with (is it muscle cells, or any other?).

It might be irrelevant, but searching Google for , or

I have found at least some publications stating something about and "dead" but so far none specifically telling about expression of Lamin A/C in daed cells:

cf: http://www.icmb.cornell.edu/faculty-staff/lammerding/lammerding-lab/pages/pubs/pdfs/LammerdingJCI2004.pdf

(Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction

Jan Lammerding,et al(2004) : and

http://jcs.biologists.org/content/114/24/4447.full.pdf

(Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and EmeryDreifuss muscular dystrophy. Wahyu Hendrati Raharjo et al, 2001).

Early embryonic cells do not express lamin A/C (cf: http://www.jci.org/articles/view/19670/pdf/render = LAMMERDING et al,2004, see also above)

Another publication (Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Tomonari Awaya et al, 2012) at http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0051638 :

Dear Chao Chi, you can alternatively use DRAQ7 to confirm whether your cells are dead/permeabilized in combination with your immunostaining. Further information is on the link shown: http://www.abcam.com/draq7%E2%84%A2-1ml-0-3mm-ab109202.html

Dear Chao,

I had a problem getting non-adherent cells for immuno-histochemistry. The trick that worked is to spin them on a glass slide (using a machine called cytospin, which is basically like a centrifugue but with slides at the botton of each well). I then proceeded with staining on the slide itself. Cells did not detach knowing that they were already fixed in formaldehye. I would suggest you couple your lamin A/C stain with another one to differentiate between live and dead cells if the distinction is not so clear morphologically.

Do you have access to FLOW cytometry? If so, this may be the way forward - collect detached and trypsinised attached cells and do a double stain for your Lam A/C and e.g. annexin V - double positives would then be an indicator of dying/dead cells and Lam positivity.

Thanks for your answers. But I only want to know if a definitely dead cell can express Lam A/C. I want use Lam A/C track the human stem cells which inject to the rat heart.

An apoptosis detection kit was used for Annexin V-FITC binding and PI staining. The cells were were harvested and washed with PBS, and then resuspended in binding buffer. My quention is can I use the PBS contained BSA to replace the binding buffer.