Hello

Kindly look at PDF files will help you

Hi,

how do you identify cells as being non-viable after 24h? Do they just not attach to the vessel, or do you use other viability markers?

Also, do I understand you right that you incubate your cells on a shaker after seeding? That is a very bad idea, as it will not allow the cells to attach. Do not unnecessarily disturb the cells, especially during the first hours after seeding.

A problem I have encountered with primary cells (however from trout, spleen and head kidney) is that the large number of erythrocytes in spleen interferes with attachment to the culture vessel. Removing them by density gradient centrifugation (e.g. Ficoll) improved attachment. Also, adding serum directly at seeding impedes attachment as well, so consider using serum-free medium for the first few hours or even the first day. Apart from those points, I use a procedure very similar to yours, and the primary cells from trout survive several weeks, however they do not notably proliferate.

You are correct on L-15 and CO2, it is designed to be used in normal air atmosphere.

Hi Patrick,

Thank you for your comments. Here are the answers to your questions:

- I think my cells are dying for two reasons: 1) I've done a few time-course experiments with the MTT and there is a major drop off in absorbance from 0 to 24 and it steadily decreases to almost the same levels as the blank by 72 hours. 2) Over the past couple of days since I posted this question I'm starting to think might be a loss of RBCs (which, like you said, I'm now thinking I need to get rid of). I initially wanted to avoid RBC lysing given that I didn't want to manipulate my suspensions anymore than I had to. I've attached a couple pictures of what my cells look like immediately after plating and then 24 hours later. Again, I'm not used to looking at attached cells, but I'm guessing those elongated ones attached whereas most of my suspension was RBCs and they have died off.

- I've eliminated the rocker. I only tried it once out of desperation!

- I agree with you, and I'm really starting to think that getting rid of RBCs is the way to go though I've also considered increasing my concentrations of FBS and glutamine to 10% and 1% in my media in an effort to promote growth and provide a more hospitable environment for cell proliferation? I think I'll try a Ficoll gradient like you suggested, but I am somewhat concerned I may not be able to visualize a distinct layer of cells given the small size of my fish (fathead minnows) and the small amount of cells I usually get (I usually pool several organs together). Do you have a concentration of Ficoll you would recommend and/or a RBC lysing solution that you have used in the past?

- Good to know I wasn't wrong about the L-15!

Thanks again for the help!

Hi,

ok, that looks very different to what I got from trout spleen in my first trys (see attached picture, 24h after seeding without RBC removal). You don't have nearly as many RBCs in there as I did (smaller, highly refractive particles, most of the particles in my picture). Also, I did not get that many large round (I assume parenchyma?) cells, and under those conditions, mostly fibroblasts did attach to the vessel.

Removing RBCs with lysis buffer is one possible approach, however I was afraid of damaging the leukocytes via osmotic stress, though it has been done sucessfully (only for analysis and again, in trout), see

https://doi.org/10.1577/1548-8667(2001)0132.0.CO;2

I decided to go for a very simplified density gradient centrifugation protocol modified from this paper Article Salmonid macrophages: Separation, in vitro culture and chara...

I used Histopaque 1.077, but classical Percoll or Ficoll should work, too, put 5 ml into a 15 ml centrifuge tube and carefully overlayed it with 3 ml cell suspension in medium (~1x10^7 cells/ml). Centrifugation in a swing bucket rotor at 400 g for 40 minutes (turn off the brake!) pelleted the RBCs at the bottom of the tube, while most leucocytes (and cell clumps) stayed on top of the Histopaque, where I retrieved them by pipetting and plated them after two washes with PBS. Also note that there is quite a bit difference in leukocyte density in between species (see the paper), so you may have to adjust the density a bit for minnow, it is possible to reduce it simply by dilution with PBS.

Do you just want to culture leukocytes, or do you specifically need them from spleen? I've had much more success with head kidney cells, and finally focused on those for my experiments, so maybe take a look at this approach, too.

Regarding the FBS, absolutely, the cells need FBS for prolonged survival, however I found that it inhibits initial attachment greatly in agreement with the literature. Therefore, I now plate the cells without FBS and add 20 % FBS and 20 µg/ml PHA the next morning. Head kidney cultures are very happy with those conditions and appear healthy even after 6 weeks with weekly medium changes.

Hi Patrick,

Thank you so much for your recommendations. They have been extremely helpful. I was able to eliminate the RBCs in my head kidney cell suspension with a lysis buffer though I will likely switch to Histopaque since I too am worried about the stress on my desired cell populations. As you recommended I plated the cells without FBS and this morning many of them appeared to have attached. I'm guessing the ones that still appear to be in suspension are lymphocytes. I will add 20% FBS shortly along with my selected mitogens and monitor viability and proliferation from there. I have also had much better luck in the kidney and will likely focus on that tissue rather than spleen. All in all, things appear to be going well! Again, thank you!