Flowchart
disaggregation of explant
Flowchart content
DISAGGREAGATION OF EXPLANT
Primary Explant Technique
Mechanical Disaggregation
Enzymatic Disaggregation
Warm Trypisination
Cold Trypisination
Finely chopped
Dissection of explant
Washed by settling
Transfer in last wash
Remove BSS
Add thin film of medium
Incubate 2 to 3 weeks
Explant outgrowth
TISSUE SAMPLE - pass through 100um mesh petri dish containing BSS
CELL + DEBRIS -
Repeat the above process through 50um mesh
MORE CELLS + DEBRIS -
Repeat & pass through
20um mesh
VIABEL CELLS + DEBRIS -
Removal of debris
DILUTE THE CELLS
INCUBATE
PRIMARY CELL CULTURE
TISSUE SAMPLE
CHOPPED TO
2 TO 3 MM
PIECES
TISSUE PIECES
WASHED WITH DBSS
TISSUE PIECES WITH WARM
TRYPSIN
AFTER 30 MIN PIECES SETTLE
SUPERNATANT
REMOVE TRYPSIN
&
CENTRIFUGE
DISPERSE CELL PELLET
IN MEDIUM
DISAGGREGATED CELLS
ARE KEPT
VIAL ON ICE
ADD FRESH TRYPSIN
& CONTINUOUS
CENTRIFUGATION
TISSUE PIECES IN A VIAL
Vial kept on ice
Soak tissue pieces in cold trypsin for 6 - 24 hr
Remove trypsin & incubate at 37 C for 20 - 30 min
Disperse cells by pippeting
Cells in medium
Count cells
Dilute in suitable medium
Incubate
Primary culture
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- Flowchart on disaggregation of explant, created by Bhuvi Suriya on 26/12/2017.
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- Bhuvi Suriya
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