I use 1.130g/mL Percoll. Then I make a 46% mixture of Percoll and X-Vivo 20 with 5% Serum.
So like 46mL Percoll, and 54mL XVIVO20+5% Serum.
And then I layer 20mL of 46% Percoll in a 50mL tube, and add 15mL of Ficoll Purified MNC(CD4,CD8,Monocytes) suspended in XV20+Serum slowly on top of the 46% percoll.
Then I centrifuge for 550g, 30 minutes, RT, brake off.
After 30 minutes, I pull it out to find that I ONLY have a middle layer. No lymphocyte pellet at the bottom.
So I collect the middle layer and run flow. I get 30% lymphocytes, and 70% monocytes.
Why aren't they separating?
Dear Nathinel Zhou!
Here is the proper separation method for monocytes.
2. Type of research
1. Generation of dendritic cells from monocytes.
2. Research on monocytes and macrophages.
3. Time required:
1. Isolation of peripheral blood mononuclear cells (PBMC) from a buffy coat on a Ficoll density gradient: 2 h.
2. Cell counting: 10 min.
3. Preparation of a cell suspension with the desired cell concentration: 15 min.
4. Preparation of the hyper- and iso-osmotic Percoll solutions: 15 min (can be done during the centrifugation or several days in advance).
5. Separation of monocytes from lymphocytes on the hyper-osmotic Percoll: 1 h.
6. Cell counting (optional): 10 min.
7. Separation of monocytes from platelets and dead cells on the iso-osmotic Percoll: 45 min.
8. Cell counting: 10 min.
The overall time required is approximately 5 h.
4. Materials
If the monocytes are to be cultivated, then the material used throughout the procedure must be sterile and endotoxin-free. 50 ml centrifuge tubes. Pasteur pipettes. plastic serological pipettes (1, 5, 10 and 25 ml). stripettor.
4.1. Chemicals and reagents RPMI medium. Ficol Percoll, product number: P1644 or P4937 (Sigma). 1.6 M NaCl solution. 1.5 M NaCl solution. Distilled or deionized water.
4.2. Special equipment Centrifuge (preferably Heraeus, Megafuge 1.0). Safety cabinet (optional, for isolation under aseptic conditions). Flow cytometer (optional, for evaluating the composition of the cell suspension).
5. Detailed procedure
5.1. Starting material.
The following protocol is most suited to the isolation of monocytes from buffy coats (BC) although it has also been applied for lymphopheresis products and can easily be adapted for smaller volumes of whole blood (see Discussion).
5.2. PBMC isolation
1. Dilute the BC with up to twice its volume of RPMI medium, most frequently to 200 ml.
2. Distribute 12.5 ml of Ficoll into each 50 ml centrifuge tube. The number of tubes depends on the final volume of the diluted BC.
3. Carefully overlay the Ficoll with 25 ml of the diluted BC.
4. Centrifuge at 950 g for 15 min with the brake off.
5. Collect the PBMC at the interface between the Ficoll and the plasma-medium layers with a Pasteur pipette and transfer the cells from two tubes into one new tube.
6. Add RPMI medium to a final volume of 45 ml.
7. Centrifuge at 350 g for 7 min. Decant the supernatant carefully and resuspend the cells in fresh RPMI medium. Combine the cells from two tubes and add RPMI medium to a final volume of 45 ml.
8. Centrifuge at 350 g for 7 min. Discard the supernatant and resuspend the cells in RPMI medium. Combine the cells from two tubes and add RPMI medium to a final volume of 25 ml.
9. Mix 20 Al of this cell suspension with 180 Al of Turk’s solution. Count the cells in a standard hemocytometer.
10. Centrifuge the suspension from Step 8 at 350 g for 7 min. Discard the supernatant and resuspend the cells at a concentration of 50 – 70 106 cells/ml in RPMI medium to give a final volume which is a multiple of 3 (i.e. 3/6/9/12/... ml).
5.3. Preparation of the Percoll solutions The Percoll solutions can be prepared in advance and kept at 4 jC until required.
5.3.1. The hyper-osmotic Percoll solution 1. For 100 ml of solution, mix 48.5 ml of Percoll, 41.5 ml of water and 10.0 ml of 1.6 M NaCl and shake vigorously.
5.3.2. The iso-osmotic Percoll solution 1. For 20 ml of solution, mix 8.3 ml of Percoll, 9.7 ml of water and 2.0 ml of 1.5 M NaCl and shake vigorously.
5.4. Separation of monocytes from lymphocytes
1. Carefully lay 3 ml of the PBMC suspension (containing 150 –200 106 cells) onto 10 ml of the hyper-osmotic Percoll solution in each centrifuge tube so that the interface between the layers is preserved.
2. Centrifuge the tubes at 580 g for 15 min with the brake off.
5.4.1. Monocyte fraction
3. Collect the cells at the interface with a Pasteur pipette. Do not collect the last 4 ml.
4. Combine the cells from up to three tubes. Add RPMI medium to a final volume of 45 ml.
5. Centrifuge at 350 g for 7 min.
6. Discard the supernatant and resuspend the cells in 3 ml of RPMI medium. The suspension is later referred to as a monocyte-enriched suspension.
7. Mix 20 Al of the cell suspension with 180 Al of trypan blue solution and count the cells in a standard hemocytometer (optional).
5.4.2. Lymphocyte fraction
8. After collecting the monocyte fraction at the interface, combine the residual sediments in one centrifuge tube. Add RPMI medium to a final volume of 45 ml.
9. Centrifuge at 350 g for 7 min. Discard the supernatant and resuspend the cells in culture medium to allow them to recover.
10. Count the cells.
5.5. Separation of monocytes from platelets and dead cells
1. In each centrifuge tube, overlay 10 ml of isoosmotic Percoll solution with 3 ml of the monocyte-enriched suspension (refer to Section 5.4.1, Step 6) (up to 200 106 cells).
2. Centrifuge at 350 g for 15 min with the brake off.
3. Collect and discard the supernatant with a Pasteur pipette, or alternatively, save it for analysis of its composition.
4. Resuspend the sediment in less than 1 ml of RPMI medium and transfer it into a new centrifuge tube. Add RPMI medium to a final volume of 45 ml.
5. Centrifuge at 350 g for 7 min. Discard the supernatant and resuspend the cells in 5 ml of culture medium.
6. Mix 20 Al of the cell suspension with 180 Al of trypan blue solution and count the cells.
To see the results, I am adding also the article.
Make the experiments and make report to me, please!
Sincerely yours!
Dr. Bratko Filipič
Email: [email protected]
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