Primary rat hepatocyte isolation

pathos

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Nov 16, 2015
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#21
Rat hepatocytes are about 18 um and RBC is about 4 um. If you can not figure out which is which then lok for the nucleus. Hepatocytes have one big one, likely more. 27 % of rat heptocytes are binuclear.



If mouse cells are not attaching then Uyou likely used to much collagenase. The collagenase btw is not one enzyme. It has thermolysin as well. Sometime refered to as neutral protease. It is required to release hepatocytes but DEADLY to them if exposed to long. Step 2 of Seglen method is saline with collagenase. make sure you digest no more than 10-12 mins. Look at the Glisson capsule if you see it looking mushy STOP and filter cells





Good luck all
 
Nov 16, 2015
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#22
hi, when we isolate splenocytes from the spleen we dont use collagenase, can i delete the collagenase etape and isolate hepatocytes without using collagenase
 

drgaju.15

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Nov 16, 2015
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#23
hi

i perfused rat liver successfuly got hepatocytes but vaibility is aroound 25 to 30% only. can any body help me how to increase viability. useing heps buffer DMEM with collagenase for perfusion, PH 7.2 @ 7ml/min.



thanks


 

Sel

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Nov 16, 2015
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#25
Dear Pathos,

I've tried to perfuse and isolate Rat's hepatocytes (we use Spraque Dawley rat) following your protocol. So far I could manage and perform complete perfusion process in 15-20 mins. But now I'm confused how the viable cells exactly look under microscope in trypan blue? I got mostly quite big cells with bright and shiny cytoplasma but with blue nuclei. Do you consider it as a live or dead cells? Do you have any pictures of rat hepatocyte that you can show me so I can compare it with mine. Or I can send you my cells picture if you allow me to pm you. What do you think about chemicals that we can use to determine the viability of the cells like MTT assay or Cell Counting Kit like CCK-8 from Fluka?

Thanks and regards
 

dimova_e

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Mar 2, 2017
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#27

hi,

curently I have experienced issue with primary mouse hepatocytes that you have been discussed years ago - big yeild, obviously high viability when count with Trypan blue and almost no cells attached. Therefore I would like to ask the same question as the guy " I'm confused how the viable heaptocytes exactly look under microscope in trypan blue? I got mostly quite big cells with bright and shiny cytoplasma but with blue nuclei. Do you consider it as a live or dead cells? "

Thank you in advance for your help!

BRs,

Elitsa
 

hmeng-1

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May 9, 2017
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#28

I have same problem with primary rat hepatocytes cultlure. I got big cells with bright and shiny cytoplasm but blue nuclei, they don't attach to plate. I think they are dead cells.