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DISAGGREGATION OF EXPLANTS
PRIMARY EXPLANT TECHNIQUE
MECHANICAL DISAGGREGATION
ENZYMATIC DISAGGREGATION
dissection of explant
finely chopped
washed by settling
trnasfer in last wash
remove BSS
add thin film of medium
incubate 2 to 3 weeks
explant outgrowth
tissue sample:pass through 100 um petri dish containing BSS
cell debris:repeat the above process with 50um mesh
more cells + debris: repeat the process with 20um mesh 20um mesh
viable cells+ debris: removal of debris
diluthe the cells
incubate
primary cell culture
tissue sample
cut in to 2 to 3 mm pieces
tissue pieces washed with dbss
warm trypsination
cold trypsination
after 30 minutes pieces settle
tissue pieces with warm trypsin
supernatant
remove trypsin and centrifuge
disperse cel pellet in medium
disaggregated cells are kept via on ice
add fresh trypsin and continuous centrifugation
tissue pieces in a vial
vial kept on ice
soak tissue pieces in cold trypsin for 6 to 24 hrs
remove trypsin and incubate @ 37 c for 20 to 30 mins
disperse cells by pippetting
cells in medium
count cells
dilute in suitable medium
incubate
primary cell culture
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- Flowchart on Untitled, created by mubeena Basheer on 28/12/2017.
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- mubeena Basheer
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