Hi all,
I isolate hemocytes of honey bee larvae and put them in culture medium. Honey bee hemocytes tend to adhere to the surface of the wells very strong so I use a thin agarose layer to weaken the adherence. This works quite fine but the cells start to aggregate like you see in picture 2. Pictue 1 shows adherent cells without agarose layer. Does anyone know what's the reason for this clumping? I use the same medium for the cells.
Best regards
Arne Kablau
Hello Arne Kablau
How do you collect the haemocytes (hemocytes)? I am asking this because insect hemocytes are quite sensitive and some insect hemocytes aggregate rapidly upon removal from the hemocoel. Collecting hemocytes in low pH or calcium buffered solutions, directly collecting the hemolymph from the pericardial sinuses, using trypsin on the hemocytes or culturing them in plasma protein cultures etc have been proposed to reduce this tendency. A detailed account of it can be found in "An Overview of Insect Hemocyte Science and its Future Application in Applied and Biomedical Fields".
Regards
I´m no expert on insect cells, however, whenever I wanted cells to grow as spheroids I add an agarose layer in order to not allow cells to attach to the plate. Maybe your layer is thicker than you think and cells are not able to grow attached and try to grow floating at the medium as spheroids.
Hi,
thanks for your reply. I will try some advices from the paper and try some different agarose layers. What concentration of agarose you use and how thick is your layer? I used 2% and in 96-well plates I put 30µl agarose/well.
I pierce the cuticle at the abdominal part of the larvae and pipette the hemolymph droplet directly into my medium in the culture plate.
Best regards
I coated with 2 ml of 2% agarose but in 60mm dish wells. It was a thick layer a didnt allow cell attachment at all.
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