Isolation of monocyte from PBMC - (Nov/17/2009 )
I isolate monocytes from PBMC by Ficoll density gradient. However, the cells are very dirty and can't adhere to dishes. I want to know the key points of isolation. Thank you!
I agree - we need a bit more info. I've never done Ficoll on adherent cells, only suspension; why would it be a problem if they don't adhere?
"dirty" = platelets? how is your ficoll procedure?
The fig showed here is cells i isolated. You can see many black spots besides cells. Induction of these cells to DCs also doesn't work. I don't know why!
1. collect PBMC from mouse's eyes
2. Mix blood with HBSS without Ca and Mg by 1:1
3. Layer mixture to Ficoll density gradient centrifugation liquid
4. 1500rpm 20'
5. remove cells from the interface to 5-6ml HBSS
6. 2000rpm 20'
7. Wash cells in HBSS for 2 times 2000rpm 20'
8. Culture cells in RPMI1640+10%FBS
CellSpecific.com on Dec 3 2009, 09:13 AM said:
1. Collect blood (be sure anticoagulant is present - EDTA or heparin)
2. Mix blood with HBSS without Ca and Mg by 1:1 (5ml total)
3. Layer mixture onto Histopaque (1:1) in a 15ml conical test-tube (10ml total).
4. Centrifuge at 1800 rpm for 10 mins
5. Collect top medium and PBMC layer together into a new test-tube and raise volume with medium (wash)
6. Centrifuge at 1000-1200 rpm for 5 mins. Repeat above step and resuspend washed cells in working medium.
Hope this helps. Let us know.
It needn't remove red cells?
The RBCs are sifted out by the Histopaque. Cell specific describes taking the plasma and PBMCs, but leaving the colloid and RBC behind. (did I understand your question correctly?)
You can separate the buffy coat from the RBCs before layering, but in small quantities this is unnecessary.
lab rat on Dec 3 2009, 11:32 AM said:
You can separate the buffy coat from the RBCs before layering, but in small quantities this is unnecessary.
But there's RBC in the monocytes everytime I got. Is it the problem of Ficoll liquid?
wind on Dec 3 2009, 11:13 AM said:
CellSpecific.com on Dec 3 2009, 09:13 AM said:
1. Collect blood (be sure anticoagulant is present - EDTA or heparin)
2. Mix blood with HBSS without Ca and Mg by 1:1 (5ml total)
3. Layer mixture onto Histopaque (1:1) in a 15ml conical test-tube (10ml total).
4. Centrifuge at 1800 rpm for 10 mins
5. Collect top medium and PBMC layer together into a new test-tube and raise volume with medium (wash)
6. Centrifuge at 1000-1200 rpm for 5 mins. Repeat above step and resuspend washed cells in working medium.
Hope this helps. Let us know.
It needn't remove red cells?
The method doesn't work. But it's Ficoll in my experiment.
The method doesn't work. But it's Ficoll in my experiment.