Dear Ivane,

I think your protocol is OK. The proportions I use are: Ficoll 3 ml and BM/PBS mixture 1:3, about 8 ml. A failure in mononuclear layer formation could be because of the centrifuge you use. It is very important to centrifuge with brakes off. Do you use Falcon tubes with conic bottom?

Hope it helps you!. Good luck!

Elena

I always used Sepax Biosafe system for automatic isolation of mononuclear cells from bone marrow but I am curious about comments for this "hand-made" procedure

Try elutriation.

I always consider for density gradient centrifugation a ratio between diluted blood/Ficoll = 4/3. Maybe you need more ficoll with 35 ml. When I work with 50 falcon tube I put 20 ml of sample over 15 ml ficoll. It is also important to set the centrifuge at room temperature.

What species are from the BM cells?

The density of cells is different from one animal species to another, and it is also different in humans.

Ficoll is used to d = 1.077 for human

And Ficoll is used between d = 1.069 and d = 1.113 for animals, especially d = 1.089 for the mouse.

Protocol looks OK. We use the same volume of Ficoll and BM-dilution. When WBC count is abnormal high we'll dilute to reduce to normal WBC. Concentration in PBS can be important, so check with the manufactorers protocol. Also it can be important that the ficoll is at roomtemperature (manufactor). We centrifuge at 700 g for 30 minutes (r=190) and start with normal speed-up, but the brake is off.

Good luck.

If possible you can also give Lymphoprep (from Axis-Shield) a try. I have excellent experience with that. But one thing I am missing is the temperature of spinning, RT (22-24) is recommended, otherwise if cooler there is a chance of MNC precipitation. You should also think about the sg of BM compared to buffy coat (the former comes with stroma), so here choice of density gradient medium is critical. as mentioned in above comments, brake should be off, no acceleration.

I also dilute BM 1:3 and layer 35ml over 12ml Ficoll (1.077) and centrifuge at 400g for 30min at RT. As expected there is more red cell contamination of the mononuclear layer from BM as there are more nucleated rbc in this tissue. The only occasions on which I have seen formation of a very dispersed mononuclear layer is when using a centrifuge where the buckets did not achieve a horizontal position during centrifugation. Check your centrifuge and if the buckets will not lie completely horizontal perhaps trying a different centrifuge would solve your problem.

kay

ივანე მოგესალმები ! პრობლემა არაა, ზურამ დამირეკოს და ჩვენთან გაჩვენებთ ყველაფერს, მაგას ყოველდღე ვაკეთებთ.

მოგესალმებით ბატონო გიორგი. დიდი მადლობა დახმარებისთვის. მე ადრე ვმუშაობდი ამ თემაზე და იზოლაციას უპრობლემოდ ვაკეთებდი. აქ ერთმა ჩემმა უცხოელმა კოლეგამ მთხოვა დამესვა ეს შეკითხვა რადგან მიუხედავად სწორი პროტოკოლისა შედეგს ვერ იღებდა. როგორც აღმოაჩინა ცენტრიფუგაში იყო პრობლემა.

გმადლობთ.

Dear Ivane,

Other than clumps that Florian mentioned (and occur quite often) BM samples contain the stromal components (the adherent fraction), so it would be a good idea to let your sample in the culture flask rest for at least one hour in the incubator. This helps most adherent cells (like stromal cells and monocytes/macrophages) attach to the flask (if you don't need them) then remove the floating fraction and proceed with Ficoll isolation. Also I think your spinning time and speed are a bit high, not sure, may be you had better reduce it, do you spin @ RT or 4 degree?, this is also an important point, to me it seems possible that 25 minutes at RT leads to cell activation and adherence, clump formation and  cell precipitation. Finally, you can also try Lymphoprep if got the chance.

Good Luck

1. Reagent and instrument requirements

● Buffer: Prepare a solution containing sterile phosphate- buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2−8 °C).

▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

● Filter (pore size 100 µm) to remove bone fragments and cell clumps.

● 15 mL Ficoll-Paque™ (ρ = 1.077 g/mL).

2. Protocol

2.1 Schematic figure of a density gradient centrifugation

2.2 Preparation of human bone marrow mononuclear cells (BM MNCs)

▲ Use fresh bone marrow only. Avoid freezing and thawing of bone marrow cells and perform all of the following steps under sterile conditions in a laminar flow hood.

1. Collect bone marrow from the upper iliac crest or the sternum by using an aspiration needle.

2. Dilute aspirated human bone marrow at a ratio of 7:1 with buffer, e.g., dilute 30 mL of bone marrow with 5 mL of buffer to a final volume of 35 mL.

3. Pass cells through a 100 µm filter to remove bone fragments and cell clumps.

▲ Note: Wet filter with buffer before use.

4. Carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque in a 50 mL conical tube.

5. Centrifuge at 445×g for 35 minutes at 20 °C in a swinging bucket rotor without brake.

6. Aspirate the upper layer leaving the mononuclear cell layer undisturbed at the interphase.

7. Carefully transfer the BM MNCs at the interphase to a new 50 mL conical tube.

8. Wash cells by adding up to 40 mL of buffer, mix gently and centrifuge at 300×g for 10 minutes at 20 °C. Carefully remove supernatant completely.

9. For removal of platelets, resuspend the cell pellet in 50 mL of buffer and centrifuge at 200×g for 10–15 minutes at 20 °C. Carefully remove the supernatant completely.

▲ Note: This step will increase the purity of the target cells in the subsequent MACS® Cell Separation.

10. Resuspend cell pellet in an appropriate amount of buffer for downstream applications. For magnetic labeling see MACS Cell Separation Reagents data sheets.

▲ Note: BM MNCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding to magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS Cell Separation