I am going to isolate monocytes from whole blood using either Histopaque or Ficoll combined with MACS. Does any of you have experiences using either Histopaque or Ficoll?
Here is the protocol that I used... hope it helps.
1. Collect blood from healthy volunteers into a EDTA containing vacutainers (or add 200ul heparin to 15 ml falcon containing 10ml of blood).
2. Dilute blood with PBS (20 ml blood + 30ml cold PBS)
3. Add 15ml histopaque to 50ml falcon. Overlay 25ml of dilute blood on to histopaque.
4. Set the centrifuge brake to “off” and spin at 1000g for 30 minutes at 20oC.
5. Remove the top most plasma layer and carefully collect buffy coat (contains the mononuclear cells) into a new falcon tube.
6. Add equal volume of PBS (or RPMI incomplete medium) and spin at 400g for 10 minutes at 20oC.
7. Resuspend the pellet in RPMI complete medium with 10% FBS and seed on appropriate plates.
Materials required:
1. Sterile syringes, Vacutainers (EDTA/Heparinized), 50ml falcons, 1XPBS, Histopaque(#...), RPMI medium, tissue culture dishes, Healthy volunteers for blood.
Note: RBC lysis - In case of huge RBC contamination during collection of buffy coat, add cold NH4Cl/EDTA solution to the pellet and incubate on ice for 5minutes and spin at 1000g for 15 minutes.(Repeat this step for 2-3 times till you get white monocyte pellet).
NH4Cl/EDTA solution: Add 4.38mg of EDTA and 1.2 gm of NH4Cl to 100ml sterile water and adjust the pH to 7.2 with HCl. Filter sterilize it using 0.2μm filter and store at 4oC.
The fresh human blood (5mL) is immediately added into 5-10 mL Di Potassium EDTA containing screw capped tubes and mixed with PBS in 1:1 in a 15mL centrifuge tube. The diluted blood is carefully layered up on equal amount over a 5 mL Ficoll or Histopaque in two other tubes and is centrifuged
I'm going to extract peripheral blood mononuclear cells(Monocytes and lymphocytes). Which material do you recommend? ficoll density gradient or histopaque?
PBMCs contain, not only monocytes and lymphocytes (B-cells, T-cells and NK cells), but also dendritic cells and plasmacytoid dendritic cells. In addition, Histopaque can be named as Ficoll Histopaque (or Ficoll-Paque or Ficoll-Hypaque). To isolate PBMCs using Ficoll (and then other cells that are contained in PBMCs), the density should be 1.077. Finally, for monocytes isolation from PBMCs, there are several methods: plastic adherence, magnetic beads and negative or positive selection, etc. You can also isolate monocytes directly from whole blood, without PBMCs isolation step.
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