I would like to observe phagocytosis with my students with earthworm coelomocytes. I extract coelomocytes with cold-shock and obtain a lot of cells. I depose on a slide a drop of the coelom liquid and leave for 15 min for ahesion of cell on slide. After that i put a drop of yeast and incubate at room temperature for 30 min. I slowly wash the slide with distilled water and stain with methylene blue for 1 min. I can observe a lot of cell like in the attach file but i cannot say if the blue things inside the cell are yeast or cytplasmic inclusion or vacuole. Is the yeast attach to the cell or to the slide?
As anyone as an experience of this model and can take a look ?
please find the protocol of phagocytosis as follows
PHAGOCYTOSIS: for plate reader absorbance
Add 100µl BFC for 10 mins @ room temp to negative controls.
Add 50µl of Zymosan/NR solution and incubate for 30 mins @ 10ºC.
Add B.F.C to all wells for 10 mins.
Spin Plate @ 700rpm for 5 mins
Pipette out supernatant
Wash cells with 100µl PBS
(wash till negative controls are clear)
Prior to last spin add series of standards of dyed zymosan to produce standard curve. (100µl per well).
Remove 100µl of PBS and resolubilise the dye by addition of 100µl of acidified ethanol To All Wells, leave for 10 mins. Shake gently @ 200rpm for 1min
Read @ 550 nm
regards
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