Instead of collagenase digestion you can simply finely chop and mechanically grind the tumor tissue in culture media and filter through 40um nylon mesh... then subject them to discontinuous Percoll gradient (we usually prefer 25-50-75 %). Ficoll 1077 is another feasible method. Unlike myeloid cells, lymphocytes can easily be separated from tumor tissue without digestion. One would like to avoid enzymatic digestion since it can also lead to shedding of cell surface markers.

Hi Jirakrit, 

I have used Ficoll (15 ml ficoll in a 50ml falcon. I overlaid this with 35ml tumor cell suspension (in RPMI medium) drop by drop so that the RPMI layer is separate. I have centrifuges for 2000 rpm for 20 min with low acceleration and deceleration. This results in a clear white band enriched in lymphocytes. The bottom of the tube is a huge mass of tumor debris.

Instead of collagenase digestion you can simply finely chop and mechanically grind the tumor tissue in culture media and filter through 40um nylon mesh... then subject them to discontinuous Percoll gradient (we usually prefer 25-50-75 %). Ficoll 1077 is another feasible method. Unlike myeloid cells, lymphocytes can easily be separated from tumor tissue without digestion. One would like to avoid enzymatic digestion since it can also lead to shedding of cell surface markers.

Thank you all, I will try to use Ficoll instead, and avoid collagenase digestion.

Thanks!

The other problem is the speed you are using. we usually use 700g for 15-18minutes.  We also use 33% percoll and we find that you dont need collagenase, as long as you are thorough with the mincing of the tissues. 

To echo off what has already been said, centrifuge speed (very slow deceleration and most of all Temperature(RM TM) is very important when using percoll or Ficoll. Also the ratio of Ficoll/percoll to your tumor cell suspension (3:1) determines the quality of the separation. 

Thank you for everybody suggestions!

But I have one more question, which is how do i know which concentration of Percoll/Ficoll is the best for my sample? I'm now very confusing about this. Moreover, in my protocol i mixed tumor cell suspension with 40% Percoll before adding 80% at the bottom of the tube. This will give the different result or not since some protocol just overlay cell suspension on the top of Percoll.

Thanks! 

In the protocols i have used previously, i was never advised to mix my cell suspension with ficoll. It is actually advised to carefully plant your cells on top of the ficoll and avoid any movement that my disrupt the layout even when spinning down. 

Below is the protcol using Ficoll-paque. I have use this protocol to isolate Lymphotes from the mouse heart and kidney

1.Using aseptic techniques, withdraw the required volume of Ficoll-Paque

PLUS.

2. Add Ficoll-Paque PLUS (3 ml) to the centrifuge tube.

3. Carefully layer the diluted blood sample (4 ml) on Ficoll-Paque PLUS. 

Blood sample

Important: When layering the sample do not mix Ficoll- Paque PLUS and the

diluted blood sample.

4. Centrifuge at 400 × g for 30-40 minutes at 18–20 °C.

5. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte

layer undisturbed at the interface . Care should be taken

not to disturb the lymphocyte layer. The upper layer of plasma, which is

essentially free of cells, may be saved for later use.

Procedure for washing the lymphocytes to remove platelets.

1. Using a clean Pasteur pipette transfer the lymphocyte layer to a clean

centrifuge tube. It is critical to remove all of the interface but a minimum

amount of Ficoll-Paque PLUS and supernatant. Removing excess FicollPaque

PLUS causes granulocyte contamination, removing excess supernatant

results in unnecessary contamination by platelets and plasma

proteins.

2. Add at least 3 volumes (6 ml) of balanced salt solution to the lymphocytes

in the test tube.

3. Suspend the cells by gently drawing them in and out of a Pasteur pipette.

4. Centrifuge at 60–100 × g for 10 minutes at 18–20 °C.

5. Remove the supernatant.

6. Suspend the lymphocytes in 6–8 ml balanced salt solution by gently drawing

them in and out of the Pasteur pipette.

7. Centrifuge at 60–100 × g for 10 minutes at 18–20 °C.

8. Remove the supernatant.

9. The lymphocytes should now be suspended in the medium appropriate to

the application.

11. Expected Value

Typical results from our laboratory.

Lymphocytes 95 ± 5% of cells present in fraction

Hope this is helpful 

Thank you very much Ndishabandi!

Your protocol is very informative for me, but my sample is tumor tissue. So, I'm not sure it will give the same result as this protocol has developed for blood sample. However, I will try it.

Thanks again!