I need human neutrophils for performing all of my experiments. It is very difficult for me to isolate neutrophils each time and perform experiments. Is their any neutrophil cell line available whose data will be accepted in research paper?
As far as I know, because of their relative ease of manipulation and their abilities, the neutrophil-like cell line (HL-60) is an acceptable system.
HL-60...that you can differentiate into neutrophils with retinoic acid treatment..........
It will be difficult for you to assert that exits carried out on cell lines are extrapolatable to primary human neutrophils. Is there a particular reason why you find undertaking a neutrophil prep every time so difficult - most neutrophil labs do this without too much trouble, and don't use HL-60s.
Charlotte,
Neutrophils are my model cell for all studies. For each experiments I need at least 35 million cells.
I am isolating neutrophils by ficoll density gradient sedimentation method. This requires lot of fresh blood. That's why I am searching for alternate for primary human neutrophils. I definitely going to replicate cell line data with primary cells.
I agree with Charlotte that differentiated HL60 cannot substitute for genuine PMN. As an alternative consider rabbit peritoneal PMN. We often used these when large numbers of PMN were needed. They are surprisingly easy to obtain and very large numbers of PMN result. Inject one adult rabbit i.p. with 100 to 500 cc of 0.1% oyster glycogen in sterile saline, wait 6-16 hrs, then harvest by draining peritoneal fluid with a 16G hypodermic needle inserted in the midline. The yield is 1 to 3e9 PMN per rabbit. If handled gently, rabbits tolerate this well, require no anesthesia, and can be re-used for the procedure after a 1-2 week rest period.
25 million neutrophils is easily obtainable from a human neutrophil prep without difficulty. We use the discontinuous plasma-Percol gradient centrifugation method, rather than Ficol, and obtain blood from a bank of healthy human volunteers (240 mL no more than once every four months) for our preps. We routinely recover 80% of the blood neutrophils, with a minimal monocyte contamination using this technique.
Although is definitely true that a cell lines can not be the same as the freshly isolated PMN from blood, the possibility to use cell line in which transfection can be performed should be considered.. In a recent publication, " Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis" the group transfect PBL 985 with amazing results... Are they better than HL-60 ???
Collins lab in UC Davis have performed gene expression studies of two known well lines that can be differentiated into neutrophil-like cells and the expression pattern suggest that PLB985 and HL60 may be very very similar in their origin , if not identical. Either cell line would work well for certain applications. These cell respond to chemotactic stimuli and will migrate comparable to primary neutrophil. However, their ability to form NETS and production of certain cytokines vary vastly. So choose your poison accordingly.
Dear Ritankar,
Thank you very much for your response. I have finished my all work using primary human blood derived neutrophils. All signaling studied have been done based upon known inhibitors.
Regards!
Durgesh
Dear Durgesh
Please, share your experience with me. I am currently working on neutrophil using percoll protocol to isolate from bone marrow. First, I find it extremely difficult to obtained enough neutrophil and what about the possibility of culturing the cells for some days for future uses??
Dear Babamale Olarewaju Abdulkareem ,
Depending upon mouse species, neutrophil number may vary. So you have to check the percentage of neutrophils, people obtained from respective mouse species.
I think you may have gone through this protocol
Article Isolation, Purification and Labeling of Mouse Bone Marrow Ne...
https://www.jove.com/video/50586/isolation-purification-labeling-mouse-bone-marrow-neutrophils-for
This protocol is very useful in isolating neutrophils from mouse bone marrow.
There is no alternative to fresh cells. Resting neutrophils only respond correctly till first 6-8 h after isolation.
Best Wishes.
Durgesh
Durgesh Pitale Thanks for your response. I hope to find it useful. Thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line