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-   -   PMN Neutrophil Cell Isolation (http://www.molecularstation.com/forum/immunology-host-pathogen-interactions/1995-pmn-neutrophil-cell-isolation.html)

kiki06 09-12-2007 12:53 PM

PMN Neutrophil Cell Isolation
 
Hello there,
I would like to isolate polymorpho neutrophil cells and culture them.

Does anyone have any tips or a protocol to help?

Thank you all :)
Kiki

dnamolecule 09-12-2007 12:58 PM

Re: PMN Neutrophil Cell Isolation Protocol
 
Hello kiki!

here is a protocol modified from the posted one by Bryan Heit:


Neutrophil isolation is easy, however they are extremely sensitive cells.

This protocol can be used with any mammalian blood (has been performed on humans, mice and cats).

Tips for Neutrophil Isolation:
  • Isolate and complete all steps at 4C as this will help to limit activation
  • Avoid jarring vibrating the isolation tubes, as this can activate the cells
  • Do NOT use glass - the cells will stick on the glass and not come off
NEUTROPHIL ISOLATION PROTOCOL

The protocol is a three step purification protocol of neutrophils:

STEP 1) Dextran sedimentation - this removes most of the RBC's. The RBC's sediment to the bottom of the tube, while the leukocytes and lymphocytes remain suspended in solution.

STEP 2) Hypotonic lysis - this removes any remaining RBC's and platlets.

STEP 3) Ficol sedimetntation - this separates mononuclear cells from neutophils. The neutrophils sink to the bottom of the ficol, mononuclear cells remain at the ficoll/isoalte interface.

You can achieve 98% pure neutrophils or better using this protocol, with little to no activation.

Steps in Detail:


1. Pipet 4 ml of ACD into a 50 ml conical tube and pour 20 ml of whole blood down the side of the tube. Gently invert the tube several times to mix. (for other volumes use 1ml ACD for every 5ml blood).

2. Pipet 12 ml (50% of blood volume) of 6% Dextran / 0.9% NaCl solution into the ACD/blood mixture and invert 18-20 times to ensure adequate mixing. Pipette the mixture into 4 15ml tubes (~10ml/tube). Let the four tubes stand at room temperature for 45min - 1hr, or until separation is complete.

3. Return to the settling blood and pipet the yellowish supernatant into a 50 ml tube. Spin at 1150 rpm for 12 minutes at 4C using a low brake.

4. Discard the supernatant and resuspend in 12 ml of the ice-cold ddH2O. Suck up and dispense repeatedly to break the pellet. After 20 seconds add add 4 ml of 0.6 M Kcl and mix several times. Dilute the solution to 50 ml with PBS. Spin at 1300 rpm for 6 minutes at 4C using a high brake.

5. Repeat Step 4 1-2 times, until no RBC's remain.

6. Discard the supernatant, and resuspend the pellet in 2.5 ml of PBS.

7. Layer the cell suspension over 3 ml of Ficoll-Hypaque (Sigma 1077) in a 15 ml tube. Spin at 1500 rpm for 30 minutes at 4C using a low brake.

8. When the cells have finished spinning suck off the supernatant, using a transfer pipet. Resuspend the pellet in 2 ml HBSS.

9. Determine the cell conentraiton using a hemocytometer, or other device.

Cells are noe ready for use. Neutrophils will remain viable suspended in HBSS for about 4 hours at 4C, and about half that at room temperature. They can also be resuspended in RPMI instead of HBSS, although this has been known to activate them.

Here's how to make the solutions used in the protocol: ___________________________ ACD: To 250ml ddH2O add:

7. 36 gm Citric Acid

14. 71 gm Sodium Citrate

9. 91 gm Dextrose

Store at 4C ___________________ 6% Dextran: To 250ml ddH2O add:

15. 00 gm Dextran (at least 100 000 MW)

2. 25 gm NaCl

Store at 4?C _______________________________

0. 6M Kcl: To 250ml ddH2O add:

11. 18 gm Kcl

Store at 4C _____________________________

:)

Modified from Bryan Heit's original protocol

liono 09-12-2007 01:02 PM

Re: PMN Neutrophil Cell Isolation
 
Hello kiki!
Hopes this help you:

Neutrophil Isolation


Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation of erythrocytes and density-gradient centrifugation of leukocytes. All procedures up to and including density-gradient centrifugation were conducted at 22° C unless stated otherwise.
  1. Blood was anticoagulated with acid-citrate-dextrose and centrifuged at 400 × g for 20 min to remove platelets.
  2. The platelet-rich plasma supernatant was removed and centrifuged at 2,000 × g for 30 min to prepare platelet-poor plasma (PPP).
  3. The platelet-depleted cell precipitate from the first centrifugation step was mixed with phosphate-buffered saline (PBS) (1:4). Dextran (Baxter, Warrington, UK) was added to a final concentration of 2% and erythrocytes were sedimented under gravity for 45 min at 15° C. The erythrocyte-depleted supernatant containing leukocytes was centrifuged at 400 × g for 10 min.
  4. The resulting leukocyte-enriched pellet was resuspended in PPP. The leukocytes were then layered over a discontinuous gradient of Percoll (4 ml of 61.5% and 4 ml of 76%) in a 15-ml polypropylene centrifugation tube.
  5. The Percoll densities were created from pure Percoll diluted with 10× PBS and PPP. Final percentages of PPP (vol/vol) in the Percoll solutions were 42.89% and 23. 97% for the 61.5% Percoll layer and 76.0% layer, respectively.
  6. The Percoll densities and layered cells were then centrifuged at 400 × g for 20 min. After centrifugation, mononuclear cells were found at the plasma-61.5% Percoll layer interface, and neutrophils were found at the 61.5-76.0% Percoll layer interface.
  7. Neutrophils were removed and washed twice in RPMI-1640. Cells were counted with a hemocytometer and resuspended at 106 cells/ml.
  8. Cells were plated with the compounds investigated in the study in RPMI-1640 containing 10% autologous-plasma-derived serum (PDS). PDS was obtained from nonanticoagulated cell-free plasma.
  9. Cell-free plasma was obtained by centrifugation of whole blood immediately after venipuncture.
  10. The cell-free plasma was incubated at 37° C and PDS was derived by removal of the protein clot.
  11. The purity of neutrophils was consistently greater than 99%.
Protocol from original of Sam Coward

Dr.Asmaaa 05-19-2009 06:24 PM

Re: PMN Neutrophil Cell Isolation
 
Hi kiki :
now i am at the beginin of my research please give your experience at this research .
many thanks.

Zagami 02-20-2010 07:35 AM

Re: PMN Neutrophil Cell Isolation
 
For to make easy job, we propose to use my experience printed in specific work : Zagami, F.: (1993) Phagocytic Index : A valid method of phagocytosis study. Agg. Med. & Chir., Vol. 11, 5, 469-478.
In materials and methods this way is experimented
Cellular yield : A discontinuous gradient of Percoll (Pharmacia) were used, starting from Percoll rendered isotonic through dissolving 0.15 M NaCl in it (solution referred to at 100 %). One part of whole blood in EDTA-K3 diluted 1:2 in NaCl 0.9 % was poured in silicone glass tube; using a syringe with a 19 G Luer needle, was poured below the level of the blood, one part of 50 % isotonic Percoll solution diluted in NaCl 0.9 %. With the same identical procedure a part of isotonic Percoll solution at 69 % was poured below the Percoll solution at 50 %. The test tube was then centrifugated for 20 minutes at 350 x g. The densities of the various isotonic Percoll solutions were determined by using graduated density (r) beads (Pharmacia) prepared by diluting each vial from 1 to 9 with 1 ml of PBS : 20 l of each contents were mixed together and place in one part of NaCl 0.9 %. The speciment was thus processed in the same identical way as the experimental of the sample. After centrifugation the granulocyte band (Figure 1) was removed with a Pasteur pipette and was subjected at two washing with PBS and each followed by a centrifugation at 400 x g for 10 minutes. Erythrocytes contamination, if present, was eliminated for osmotic lysis resuspending pellet with one part of NaCl 0.9 % and adding three parts of distilled water. The suspension was delicately agitated for 30 seconds and one part of NaCl 3.5 % was added in order to stop the reaction. After centrifugation at 400 x g the suspension was subjects to washing with PBS, resuspended in PBS and the recuperated cells were analyzed.
If you desire to receive the image (Figure 1) gotten after centrifugation on discontinuous gradient of Percoll, contact to me at email : [Only registered and activated users can see links. Click Here To Register...]

arvadia.pratik 05-26-2010 04:25 AM

Re: PMN Neutrophil Cell Isolation
 
hey, i am starting my new project with neutrophil isolation and neutrophil mRNA isolation. does any of you guys have worked with polylympholyte media used in neutrophil isolation. i use human serum albumin to culture neutrophil. moreover does any body used fMLP or PMA, i m kind of optimising the reactions in neutrophils.

tahirktk25 04-18-2011 12:16 AM

Re: PMN Neutrophil Cell Isolation
 
Hello every body!

I am working on human neutrophil proteomics and I want to extract neutrophil proteins for iTRAQ analysis.

1- Could anybody help me to extract proteins for iTRAQ LC-MS/MS analysis?

2- I need a protocol to culture human neutrophil...

egt 10-16-2012 10:43 AM

Re: PMN Neutrophil Cell Isolation
 
Hello!

I want to isolation neuthropil to analize his histones, but I've seen some degradation in H3 and H4. How I could avoid degradation of histones during the isolation?

I use protease inhibitors during all the process but is not enought

Thank you.

Zagami 07-24-2014 04:38 PM

Re: PMN Neutrophil Cell Isolation
 
Confirmations on my method go to the address
[Only registered and activated users can see links. Click Here To Register...]


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