Hi.
I am currently trying to isolate Neutrophils from mouse blood. I am using histopaque 1077 and 1119. I have followed the protocol set out by Sigma, which involves layering histopaque 1119 and 1077, followed by the whole blood. I spined it for 30 min at 700g.
The PBMC layer is evident, but weren't able to see the neutrophil layer (see attached image). The RBC don't seem to sediment. I had tried the same protocol using human blood and it worked. I'm not sure why it isn't working with the mice blood.
Can anyone give any tips or recommendations on isolating neutrophils from mouse blood.
Thank you:)
Hello Priyanka Dhanraj
I would suggest another protocol. Instead of using whole blood, collect the white buffy coat and layer it on Histopaque-based density gradient. You may follow the method provided below.
1. Centrifuge mice blood for 10 min at 200 × g, room temperature, in a benchtop centrifuge.
2. Collect the whitish buffy coat layer which is the layer of WBCs that appears between plasma and red blood cells.
3. Count the cells in buffy coat layer using a hemacytometer.
4. Resuspend 3–5 × 10^7 buffy coat cells in 1 ml of 1 × PBS.
5. Add 3ml Histopaque 1119 to a 15-ml tube.
6. Gently pipet 3ml Histopaque 1077 on top of the Histopaque 1119 layer.
(Kindly note: Histopaque 1119 and Histopaque 1077 should be brought to room temperature before use. Also, exercise caution to avoid disturbing the interface between Histopaque 1119 and Histopaque 1077).
7. Carefully layer 1ml of cell suspension (which you have prepared in step 4) over the upper gradient, adding the cell suspension slowly along the side of the tube so as not to break the gradient surface.
8. Centrifuge for 30 min at 700 × g, room temperature, without using a brake.
9. Using a pipet, collect neutrophils from the interface of the Histopaque 1077 and 1119 layers and transfer to 15ml tube.
10. Wash the cells three times, each time by adding 10ml of 1 × PBS, centrifuging for 5min at 200 × g, 4°C, and then removing the supernatant.
11. Determine the cell purity and number of viable cells as follows.
a. Cell purity
1. Take 20μl aliquot of cell suspension and add 180μl of medium containing 50%FBS.
2. Centrifuge the sample onto slide for 5 min at 1000 rpm in a Cytospin cytocentrifuge. Stain with Diff-Quik staining solution. Examine the cells with conventional light microscopy at 400 × to evaluate neutrophil purity.The cells showing fine granules and multilobed nuclei are counted as neutrophils.
b. Viability
Count the number of viable cells by trypan blue dye exclusion.
Best.
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