Problem about under agarose migration assay to detect neutrophil migration

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Jim Szeto
Jim Szeto's picture
Problem about under agarose migration assay to detect neutrophil migration

Hi, i am carrying a research to investigate about identifying the possible chemoattractant to induce neutrophil chemotaxis. And i choose the under agarose migration assay to take video about neutrophil migration.
 
However, during the incubation of the assay ( 37 degree celius, 5% carbon dioxide and 2 hours incubation), all neutrophil just start to move to the edges within the wells in the first half hours and stop the migration under agarose. It seems not the problem of chemoattractant seems the neutrophil migrate form the middlle space of the wells to the edge and then like blocking by something else. And the problem still exist as i lower the agarose concentration form 1.2 % to 0.5 %. Is there any specific type of agarose that have to been used?
 
Does anyone have any idea what the problem is?
Are there anyone who have set this assay before and would you give some suggestions to me?
THX~