My steps:
a) Obtain some small tissues containing target cells with a smooth entry needle tip.
b) Tissues are transferred into PronaseE ACSF solution (2mg/ml, sigma)for 15min in 37℃ water bath.
c) Then gently mix cells by pipetting up and down 10 – 15 times and filter cells using a 70um strainer.
d) Centrifuge cells at 400 rcf for 3 min.
e) Remove supernatant without disrupting the cell pellet and add 200ul ACSF with 15% BSA to the tube. Gently pipette mix 5 times.
f) Centrifuge cells at 800 rcf for 5 min.
g) Remove supernatant without disrupting the cell pellet. Add 200ul ACSF without BSA to achieve the target cell concentration.
h) Centrifuge cells at 300 rcf for 3 min.
i) Remove supernatant without disrupting the cell pellet. Add200ul ACSF without BSA to achieve the target cell concentration.
j) microscopic examination
But I got the bad cell suspension everytime and I have no idea with it.
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