One thing I noticed when I was separating hepatocytes (via a different method) was that if I put the cells into a cold environment, as in ice, the cells would die....in the process the nuclei separated from the cytoplasm.Therefore, my question is this: is it possible that these are fractionated pieces of the cell you are seeing? The nuclei are quite large as I remember. P. Echeagaray CAWAGN@MONSANTO.COM wrote: > > Fellow flowers: > > I am working with mouse hepatocytes from both CD-1 and p53 knockout > mice. We are obtaining hepatocytes by collagenase perfusion followed > by percoll gradient separation. After separation, I should have a > pretty pure hepatocyte population. When I look at the cells on > Forward vs. Side scatter, there appears to be approximately three > separate populations. I am not sure if they are all hepatocytes that > differ in size, it there are clumps of cells, or if there is > contamination of other cell types in my cell prep. > > I need any information that any of you might have pertaining to > markers that would be hepatocyte specific, and any > fixation/permeabilization methods that might work. > > I tried to use anti-cathepsin D and anti-transferrin (rabbit > polyclonal) from Dako, but didn't see any fluorescence above > background. This may have been due to my method of fixation and > permeabilization. I used 1% paraformaldehyde/20fg/ml lysophosphatidly > choline (2 min. RTx), followed by absolute methanol (10 min. on ice), > followed by 0.1% lysophosphatidly choline (5 min. on ice). > > For staining, I blocked with Caltag anti-FC receptor (MM7200) for 30 > min. at on ice, washed 2 x with 10% heat inactivated rabbit > serum/Dulbecco's Phosphate Buffered Saline without Calcium,Magnesium, > followed by 30 min. incubation on ice with a 1:20 dilution of 1x > antibody. The antibodies are diluted in 10% heat inactivated rabbit > serum/Dulbecco's Phosphate Buffered Saline without Calcium,Magnesium. > > After incubation, the cells are washed 2 x with the rabbit/DPBS and > then blocked again with anti-FC receptor as above. The cells were > then incubated with a 1:40 dilution of Santa Cruz goat anti-rabbit > Fitc for 30 min. on ice. > > After incubation, the cells are washed 3x with rabbit/DPBS, and fixed > in 1% paraformaldehyde until analysis. > > I am not sure if the antigens may have been washed out of the cells by > my fixation method. I am also not completely sure if the antigens are > cytoplasmic , or if they are nuclear. > > What I hope to do is identify the hepatocyte population fluorescently, > and then backgate onto the FSC vs. SSC plot. > > Thanks to those of you who have already helped me a great deal and in > advance to those who will, > > Connie Wagner > Monsanto Environmental Health Laboratory > 645 S. Newstead Ave. > St. Louis, MO > email address: cawagn@ccmail.monsanto.com > phone: (314) 694-7971 > fax: (314) 694-7938
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