Isolation of macrophages/monocytes and neutrophils?

Protocol

The density gradient separation method is used to isolate human neutrophils from whole blood using a mixture of sodium metrizoate and Dextran 500. This method is based on the mononuclear leukocyte separation method by Boyum (1968) which was modified for neutrophil separation by Ferrante and Thong (1980).

After collection from a donor, whole blood may be anticoagulated with EDTA, citrate, or heparin. Since they are short-lived, neutrophils should be used within 2-4 hours of collection. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended to desired concentration.

Neutrophil Isolation Protocol

Bring all reagents to room temperature.

Collect 5.0 ml of neutrophil isolation media in centrifuge tube. Carefully layer 5.0 ml of blood over the separation media. Perform this step slowly and carefully, and with the pipette tip close to the surface of the media to avoid mixing the blood and the media.

Centrifuge at 500 RCF for 35 min at 20-25°C. The blood should separate out into 6 distinct bands: plasma, monocytes, isolation media, neutrophils, more isolation media, and the red blood cell pellet. If these bands are not clear, the separation process was not clean and will need to be repeated.

Carefully remove the top three layers (plasma, monocytes, and isolation media) using a pipette. Dispose of these layers.

Carefully pipette the layer of neutrophils and all of the isolation media beneath the neutrophils. Place the solution into a clean centrifuge tube.

Dilute the neutrophil solution to 10 ml with HBSS without Ca2+/Mg2+. Invert the tube a few times to suspend the cells.

Centrifuge the neutrophil solution at 350 RCF for 10 minutes. A red pellet should be present at the bottom of the tube, containing neutrophils and residual red blood cells (RBCs). Remove the supernatant with a pipette carefully so that the pellet is not disturbed.

To lyse the residual RBCs, add 2 ml Red Cell Lysis Buffer to the tube. To resuspend the pellet, vortex the vial at a setting of 3-4. Avoid increasing the vortex setting above 4, since this may cause the neutrophils to activate. It may be necessary to vortex for several seconds, or to "pulse" the vortex to dissolve the pellet.

Centrifuge the tube at 250 RCF for 5 min. Remove the supernatant with pipette. Repeat the lysing process if required.

Add 500 μl HBSS without Ca2+/Mg2+ to each tube. Again, vortex to resuspend the pellet at a setting of 3-4. Dilute to 10 ml with HBSS without Ca2+/Mg2+.

Centrifuge the tubes at 250 RCF for 5 min. Aspirate the supernatant and discard.

Resuspend the pellet in 250 μl HBSS/HSA Solution (2% HSA). Cells may then be counted and adjusted to desired concentration.

Which is the most efficient etching solution for nickel based alloys?

Which is the most convenient manufacturing software for Additive Manufacturing?

Which is the best place in the world for the plant biotechnology research?

Which SAR dataset to use for generating coherency matrix and performing target decomposition (Description) ?

How does one access the Cranet survey data on HRM practices?

Genome-wide gene identification protocol ?

Can the problems of overfitting be solved without cross validation?

Research Gate Does it help researches to publicize their scientific work ?

How to Study a proton NMR of Graphene prepared by Electrochemical Exfoliation technique?

How to classify and recognize unseen image class in document sorting?

Issues with Permeabilization during Flow Cytometry?

What could be some reasons why unstimulated monocytes are getting activated?

Recommendations on homogenizers for RNA isolation?

How to improve RNA isolation?

What might be the reason for obtaining two bands in plant genomic DNA extraction?

Looking for advices and tips to improve the staining and the gating strategy in human blood?

FDC and FRC cell populations in mouse spleen?

Macrophages in culture not growing/ dying or detaching . Why ?

Rat Macrophage Markers for Flow Cytometry?

Can anyone help me with doing resorption pit assay on biomaterials for 28 days?