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    • #17720
      Thom
      Participant

      Hello everyone,

      A simple question, but i’m fed up with this.

      I work with hemocytes of a invertebrates. Many articles perform centrifugation steps after collecting the hemolymph, whether in eppenddorf, whether directly on a 96-well plate. Theses cells are circulating blood cells but display quite good adherence.

      Here is my problem: after centrifugation ( I tried 5 min at 300g, 10 min at 300g and 5 min at 1000g, in eppendorf and with 96-well plates, flat-bottom and round bottom). I took the supernatant off and tried to resuspend the cells with PBS and then performed my classical viability protocol… But the cells have gone… These velocities are the one used in articles… and i can’t increase the velocity of centrifugation because i want my cells in viable status… I can’t figure out where my trouble comes from.

      Apparently it is not enough to have pellet that will not detach after a PBS-washing step … but many article do not report that, so the problem is from protocol.

      Any ideas??

      Many thanks in advance,

    • #115010
      jonmoulton
      Participant

      Are you resuspending in a PBS solution isotonic to the cells? Lysis could explain why the cells aren’t there. You could try slightly increasing the tonicity of the PBS – better to have crenated cells than lysed cells.

    • #115011
      Thom
      Participant

      Yes my PBS is isotonic to my cells (i guess so because many authors used it toward such cells).
      Even if the cells were lysed, i expect them to be "pelleted" to the bottom of the wells and so when i wash, the cells should stay sticked, unless i resuspend with PBS containing EDTA or heparin… Is it correct?
      When i take off the medium after centrifugation, and then add PBS are the cell straightforward resuspended or can I take the new added fresh PBS off again until I really resuspend cells by pipetting up and down thoroughly?

      Many thanks again,

    • #115012
      jonmoulton
      Participant

      Before you wash the cells, can you see any pellet? Wash gently, add your final PBS and pipet to resuspend. Your protocol sounds good to me.

    • #115013
      Thom
      Participant

      I can’t see any pellet, BUT today it tried again, after 24h of incubation (cells have time to adhere), and centrifugation at 400 for 5 min, I gently took off the medium and resuspend the cells by pipetting thoroughly up/down: the viability worked well. However, that time i skiped the washing step (and wait 24h…), which can be annoying when working with different dyes… I will try to wash gently and gently take off the fresh-washing-PBS before really resuspending and i’ll see…

      I guess the pellet can’t be viewed (with eye’s limit of detection..) at this amount of cells…

      Thank you for the comments,

    • #115016
      JackBean
      Participant

      I’m not expert in animal biology, but I’d say you do not need EDTA or heparin for resuspension, since the cells are pelleted because of centrifugation, not because they grew there.
      What about trying some Western or other protein detection after the resuspension to see if there is anything at all? If you’ll get some signal, you get the cells pelleted, they are just not viable. If you do not get any signal, you have problem with your centrifugation.

      Teo things to consider – density and viscosity of your sample (is that only the lymph or do you add anything?) and amount of your sample in the tube/well.

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