12 replies to this topic

[wind]

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!

[CellSpecific.com]

Do check for cell viability? What do you mean by "dirty"? Perhaps giving a bit more information about your isolation method might allow us to see what you're doing that could be improved.

[aimikins]

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?

[hoshi]

"dirty" = platelets? how is your ficoll procedure?

[wind]

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!

Attached File(s)

•  Image_2713.jpg (135.23K)
  Number of downloads: 27

[CellSpecific.com]

I'm repeating hoshi's question: Can you tell us (step-by-step) how you isolated the cells.

[wind]

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]

Try this:

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.

Edited by CellSpecific.com, 02 December 2009 - 05:15 PM.

[wind]

CellSpecific.com, on Dec 3 2009, 09:13 AM, said:

Try this:

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?

[lab rat]

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.

Edited by lab rat, 02 December 2009 - 07:34 PM.

[wind]

lab rat, on Dec 3 2009, 11:32 AM, said:

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.

But there's RBC in the monocytes everytime I got. Is it the problem of Ficoll liquid?

[wind]

wind, on Dec 3 2009, 11:13 AM, said:

CellSpecific.com, on Dec 3 2009, 09:13 AM, said:

Try this:

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.

[Astarte Biologics]

The method doesn't work. But it's Ficoll in my experiment.
[/quote]

Are you using Ficoll-Paque? Or Histopaque or Lymphoprep? These are all trade names for the gradient material used to isolate PBMC. There is also just Ficoll - a sucrose polymer- trademark of Pharmacia. Sorry to ask such a basic point but assuming things leaves a lot out.

If you are trying to isolate mouse DC there are other methods rather than using peripheral blood.

You may want to check what cell types you have in the initial prep. Human PBMC contain 10-15% monocytes but mouse WBC are a different range of cell types. Also, the density of mouse lymphs is bit higher than human so if you use the most common formulation of Ficoll-Paque you will lose some cells in the pellet.

More details please and you'll get more specific input. Hope we can help.