I am using a polymorphprep solution for isolation of neutrophils from human blood. I am using all the protocols and instructions provided by Axis shield but not getting a layer of neutrophil but rather a layer related to monocyte is visible every time.
I don't know your specific steps, but it is very important first of all to make sure that the blood is layered on top of the polymorphprep (no mixing) for the gradient layers to come out properly. We spin our samples at 500g for 45 minutes. Then you will see a top layer of yellow plasma with a layer of lymphocytes underneath. Under that there is some clear fluid and then a cloudy layer that is a bit difficult to see, but that's where the neutrophils are. I usually aspirate the top layers off and then use a 200-1000 pipette to extract the granulocytes and transfer to another tube. Fill that with isotonic buffer (HBSS is what we use) and spin. Then you will have a neutrophil pellet.
I don't know your specific steps, but it is very important first of all to make sure that the blood is layered on top of the polymorphprep (no mixing) for the gradient layers to come out properly. We spin our samples at 500g for 45 minutes. Then you will see a top layer of yellow plasma with a layer of lymphocytes underneath. Under that there is some clear fluid and then a cloudy layer that is a bit difficult to see, but that's where the neutrophils are. I usually aspirate the top layers off and then use a 200-1000 pipette to extract the granulocytes and transfer to another tube. Fill that with isotonic buffer (HBSS is what we use) and spin. Then you will have a neutrophil pellet.
Layering and RT temperatures and no brake during the centrifugation are very important.
I have several methods for layering:
1) you fill a falcon with half volume of polymorphprep and you incline the falcon allowing a line of liquid to come at the top end. Then you take your sample with a pipet boy and put the end of the pipet at the top of this falcon, then you pour the sample very very slowly and it will run towards the polymorphoprep without mixing with it. While you are pouring you incline (SLOWLY) the falcon in the standing position while you are inclining the pipette as well. You should keep the pipette on the falcon wall all the time. In this way you will have an higher yield, thus resulting a more clear ring of neutrophils.
2) you put your sample in an universal tube. Then you put a glass-made pasteur at the end of the universal tube (in the middle of it). Then you fill of polymorphprep the pasteur from the end (of course not in one-go). The liquid will go slowly exactly under your sample and it will not mix with it.
thank you Alden Moss and Elisa Balmas for your nice suggestions, here i am sending the protocol currently using for isolation of neutrophils.
1. collected fresh venous 5ml blood in NaEDTA tubes.
2. Then layered whole 5ml blood above the 5ml poymorphprep solution in a 15ml falcon tube ( there was a clear separation in between)
3. then centrifuged at 505g for 30 mint at 20 o C
4. but i am only getting upper band of monocyte and unable to find any neutrophil band.
plz suggest me and kindly send me whole modified protocol for this
thank you
When you are interested in pure neutrophils without contaminating eosinophils the fastest way is our new kit. I attached the poster we presented at the ICI 2013 in Milan.
Hope this helps,
Gregor
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology