Perhaps it helps to have the exact protocol:

1) Separate the monocytes from blood with Ficoll-plaque.

2) Resuspend the RBC/Neutrophil pellet in 3% Dextran/9%NaCl. Let it sit 20 minutes, then remove the upper plasma layer and centrifuge that for 10 min at 250g.

**At this point the cells don't seem to pellet.

3) Add 0.2% NaCl to lyse residual RBC, then 1.6% (total time 28 seconds), and then 0.9% NaCl, then centrifuge at 10m/250g.

Can I just centrifuge at higher G, to get the cells to pellet?

Are a lot of cells in the interface?

It will depend on the species since densitiy of neutrophils will vary. Here a study in pigs in which a discontinuous Percoll gradient was used

Article The effect of β-glucans on porcine leukocytes

Hi Michael,

Even with variable density, I believe most of neutrophils should pellet at 250g.

Have you actually checked that you have neutrophils in the upper layer following the dextran sedimentation, and that they are not in the bottom layer with the RBC? We use 6% dextran (T500) in our lab; with 3%, there may be the possibility that neutrophils will sediment with the RBC? Hope that helps.

Best Wishes

Marie

Sounds like your percent dextran is too high. I use 2%. To make the dextran solution, add 20 grams to about 800ml ddH2O and 9 grams NaCl. Let it dissolve, then bring the volume up to 1000ml.

Hi Michael

Sorry can not help you,we use a zebrafish transgenic line that express GFP only in neutrophils and isolate them by Facs

Regards

Carmen

Hi Michael,

3% Dextran in 0.9% NaCl is minimal conc. used for neutrophil isolation. Try to spin the cells at 800 g for 15 min without break at 4C after 20 min incubation. Perform all the experiments at 4C. Even if you see more RBC, lyse them in endotoxin free water for 30 sec. and immediately resuspend in saline followed by spin at 800g for 5 min. You can repeat this lyssi step couple of times till you see a white cell pellets.

Good Luck

Michael.. Its Dextran first, THEN Ficoll cushion. Check published protocols, for example in PMID 17643350.

Hey everyone, thanks for the assistance. It worked perfectly today.

Hi Michael. Can you try with Histopaque 1119? I use it.

we use the following protocol for isolation of human neutrophils (adapted from Neutrophils Methods and Protocols):

- after Ficoll-plaque isolation: add 20mls of HBSS to the red cell pellet

- add 20mls of 3% Dextran (Dextran from Leuconostoc, average mol wt 425,000-575,000) in HBSS and invert 10 times

- let sit for 18min

- transfer upper layer (neutrophils and erythrocytes) into a new tube and centrifuge at 500xg for 5min

- discard supernatant and lyse erythrocytes with sterile water for 28 seconds

- add 1.8% NaCl immediately, mix and centrifuge at 500xg for 5min

isolating neutrophils without activation is difficult. For mouse bone marrow, the Stem Cell negative selection kit is excellent. For human neutrophils, Percoll is better than Ficoll. Miltenyi CD66b kit activates the neutrophils, too. The Percoll method is described in PMID: 2467707

The best protocol we have used to obtain/ isolate non- activated human neutrophils is using non-continuous Percoll gradiente. You can try :

Isolation of Neutrophils (PMNs).

This method uses discontinuous Percoll gradient to obtain a neutrophil rich mixture polymorphonuclear granulocytes (i.e. neutrophils, eosinophils and basophils). This prepation yields over 98% purity and 97% viability. Of the granulocytes, there is under 5% eosinophils, and under 1% basohphils.

1. Prepare 72, 63, 54, 45% (v/v) Percoll solutions – in Hank’s (for 3 gradients prepare 7 mL/ each solution).

72%: Percoll 5.04mL + Hank’s 1x 1.4 mL + Hank’s 10x 560µL

63%: Percoll 4.41mL + Hank’s1x 2.1mL + Hank’s 10x 490µL

54%: Percoll 3.78mL + Hank’s 1x 2.8mL + Hank’s 10x 420µL

45%: Percoll 3.15mL + Hank’s 1x 3.5mL + Hank’s 10x 350µL

2. Carefully pipette 2mL of each Percoll solution (starting with 72%) into a 15ml polystyrene centrifuge tube. Take care not to introduce air bubbles. Carefully overlay the 72% layer with the 63% layer. Do the same until 45% layer.

3. Dispense 15 mL freshly drawn venous blood into a 15ml centrifuge tube containing 350µL 5% (w/v) EDTA. Use a clear sided tube (Such as polystyrene).

4. Carefully overlay 2mL of blood onto the prepared discontinuous gradient in step 2 down the wall of the tube.

5. Centrifuge at 500g for 35 minutes at room temperature, 2 brake.

4. Aspirate and discard the top layer rich plasma/platelet and the second layer rich monocyte/lynphocyte. (If required, monoctyes and lymphocytes can be harvested from the interface of the 54% layer).

5. Harvest the PMN from the 63% / 72% boundary (2mL approximately).

6. Transfer PMN fraction into 15mL centrifuge tube containing 10mL of Hank’s solution and centrifuge at 300g for 10 minutes. Discard supernatant.

7. Gently resuspend cell pellet in 7mL 0.2% (w/v) sodium chloride (to lyse the red cells) and then add more 7mL 1.6% (w/v) sodium chloride. Gently mix. (Avoid vigorous resuspension as it results in PMN activation)

8. Centrifuge at 300g for 10 minutes. Discard supernatant.

9. Resuspend PMNs in cell media (RPMI).

10. The PMNs are now ready for use.

Hi I like Dr Kasper response. My experience with Percoll and neutrophils is not very good, it activates them. So if you are going to perform the separation keep in mind that you should use EDTA 0.5 mM and 0.5 % gelatin in PBS so they will not agregate. Good luck

Hi Michael

You will get better results with Percoll instead of Ficoll. Percoll must be without cristals, since these would be phagocytosed like Ficoll.

See: Alstaedt J., Kirchner H. and Rink L. (1996): Cytokine production of neutrophils is limited to IL-8; Immunology., 89:563-568

Depending on the purity you can reduce the protocoll to one step Percoll. It is favourable to use HAES instead of Dextran.

Best regards

Lothar

Has anybody used the Miltenyi kit for magnetic-bead separation for bone marrow neutrophils? (similar to the StemCell kit). Any insight regarding if these kits elicit some activation? I wanted to try using the Histopaque-1119 and -1077 method described in JoVE (http://www.jove.com/video/50586/isolation-purification-labeling-mouse-bone-marrow-neutrophils-for), but they are out of stock until the end of this month. Thanks everybody in advance!

Have you got a good protocol?

I may suggest that after collecting the upper layer from dextran sedimentation step, mix the layer with 2-3 times volume of HBSS (1X) to dilute the concentration of dextran before centrifugation.

I am using a density gradient commercially available and it's working fine but you select all PMN cells present in the blood and at the same time you do not loose the MN fraction. It is working fine (80% pure) but more the blood, better the separation; I use 20 ml  

http://www.axis-shield-density-gradient-media.com/Pmpp.pdf

The method suggested by Christina Barja-Fidalgo it's more accurate but the principle is the same,    isn't it ????