Isolation of Lymphocytes, Peripheral Blood Mononuclear Cells, PBMC

Protocol 1

1. Mix 20 mL fresh blood or EDTA/citric acid treated blood (~2 10^7 cells) of each donor with 20 mL PBS.

2. Fill 10 mL Lymphoprep into a 50 mL polypropylen tube. Carefully overlay Lymphoprep with 40 mL of the diluted blood using a plastic pipette.
Note: Human blood samples are potentially infectious (HIV, hepatitis, etc.)!

3. Centrifuge the blood with 800 xg for 20 min at RT (without brake!). 

H-1000B https://www.sciencegateway.org/tools/rotor.htm:  H-1000B (buckets),  400 xg -->1500 rpm; 800 xg  --> 2000 rpm

4. The lymphocytes form an opaque phase separation layer between the Lympho- prep and the medium, whereas the erythrocytes and granulocytes will be pel- leted. Carefully aspirate the lymphocytes using a plastic pipette and transfer to a new 50 mL polypropylen tube.

5. Fill up with 50 mL PBS and pellet the lymphocytes with 250 xg for 10 min at RT. Discard the supernatant (be careful, the lymphocyte pellet is not solid).

6. Repeat this washing step to remove most of the thrombocytes.

7. Resuspend the lymphocytes pellet in the supplied extraction buffer of them RNA isolation kit according to the manufacturers’ instructions or use 0.5 mL Trizol for total RNA isolation. Both methods, mRNA or total RNA isolation, work well. After resuspension using the mRNA extraction buffer or Trizol, the RNA pellet can be stored at  -80 ^oC.

Home-made Ficoll-Paque

Ficoll-Paque of density 1.077g/ml: To 100ml water, add 5.7g Ficoll and 9g sodium diatrizoate.

Protocol 2

The separation can be done in either 15 or 50 ml conical tubes.

1. Aliquot 15 ml of Ficoll-Paque PLUS or Histopaque-1077 for every 15 ml of peripheral blood to be processed.

Let Ficoll-Paque PLUS or Histopaque-1077 warm to room temperature as density is temperature-dependent.

2. Mix 15 ml of blood with 15 ml of a balanced salt solution, normally PBS.

3. Overlay the mixed blood solution on top of the Ficoll-Paque PLUS or Histopaque-1077.

Ratio should be 2 ml of the mixed blood + PBS solution for every 1 ml of Ficoll-Paque PLUS or Histopaque-1077.

4. Centrifuge at 400 x g for 35 minutes at room temperature. 

H-1000B https://www.sciencegateway.org/tools/rotor.htm:  H-1000B (buckets),  400 xg -->1500 rpm; 800 xg  --> 2000 rpm

During this step, the granulocytes, platelets and RBCs pellet to the bottom of the tube and the PBMC float over the Ficoll-Paque PLUS or Histopaque-1077.

5. Carefully aspirate the PBMC from the Ficoll-plasma interface.

6. Wash the PBMC with PBS at 300 x g at 4 oC twice. 

Protocol 3 Ficoll® density gradient separation of PBMC

1. Transfer 10 mL of blood from the collection vial to a 50 mL tube.

2. Add an equal volume (10 ml) of PBS 

• Note: Diluting blood reduces the degree of RBC aggregation as well as freeing trapped PBMC. If left undiluted, trapped PBMC sediment with erythrocytes, reducing yield.

3. Add 15 mL Ficoll® (GE Healthcare) to a second 50 mL tube.

4. Carefully layer the diluted blood over the Ficoll®.

• Note: The diluted blood is added to the gradient by gently pipeting onto the separation medium with the tube held at an angle. To obtain good separation, it is paramount that clean separation of the blood and Ficoll® layers is maintained prior to centrifugation

5. Centrifuge without the brake applied at 400 g x 30 min at 18-24°C.

• Note: Higher temperatures (37°C) enhance RBC aggregation reducing yield while lower temperatures (4°C) inhibit aggregation, decreasing purity. We recommend centrifuging at 18-24°C.

6. Carefully remove the tubes from the centrifuge so as not disturb the layering.

7. Draw off the upper plasma layer being careful not to disturb the lower PBMC interface.

8. Remove the PBMC layer and transfer to a new 50 mL tube. The volume recovered should be approximately 10-12 mL.
• Note: Attempt to remove the entire interface while minimizing the amount of Ficoll® or remaining plasma layer. Excess Ficoll® or plasma recovery will result in contamination by granulocytes or platelets and plasma proteins, respectively.

9. Wash PBMC fraction using ~3 volumes of PBS. Centrifuge at 100 g x 10 min at 18-24°C.

10. Decant the supernatant. Resuspend the pellet in 5 mL PBS. Add PBS to 50 mL and repeat wash step.
• Optional: The wash step can be repeated once more

Protocol 3

This protocol is optimized for the preparation of PBMCs from human blood. For the preparation of PBMCs from rhesus monkey (Macaca mulatta) or cynomolgus monkey (M. fascicularis) blood, it is recommended to use 96% Ficoll-Paque". • The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)). 

1. Reagent and instrument requirements

Buffer: Prepare a solution containing 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). 

15 mL of Ficoll-Paque" (p = 1.077 g/mL). 
For protocol 2.3: Leucosep® Tube (# 227 290, Greiner Bio-One GmbH). 

2. Protocols 

2.1 Schematic figure of a density gradient centrifugation 

2.2 Isolation of peripheral blood mononuclear cells (PBMCs) using Ficoll-Paque-

 This protocol is optimized for the preparation of PBMCs from human blood. For the preparation of PBMCs from rhesus monkey (Macaca mulatta) or cynomolgus monkey (M. fascicularis) blood, it is recommended to use 96% Ficoll-Paque". 

The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)). 

Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation 
1. Dilute cells with 2-4x the volume of buffer.

• Note: The more diluted the blood sample, the better the purity of the mononuclear cells. 

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

3. Centrifuge at 400xg for 30-40 minutes at 20 °C in a swinging-bucket rotor without brake. 

4. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.

5. Carefully transfer the mononuclear cell layer to a new 50 mL conical tube. 
6. Fill the conical tube with buffer, mix, and centrifuge at 300xg for 10 minutes at 20 °C. Carefully remove supernatant completely.

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

8. Repeat step 7. Most ofthe platelets will remain in the supernatant upon centrifugation at 200xg.

9. Resuspend cell pellet in an appropriate amount of buffer and proceed to magnetic labeling. For details see MACS Cell Separation Reagents data sheets. •

Note: PBMCs 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 Reagents data sheets. 

Protocol 4 Isolation of PBMCs using Ficoll-Paque and a leucosep Tube

This protocol is optimized for the preparation of PBMCs from human blood. For the preparation of PBMCs from rhesus monkey (Macaca mulatta) or cynomolgus monkey (M. fascicularis) blood, it is recommended to use 96% Ficoll-Paque". • The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)).

1. Dilute cells with 2-4x the volume of buffer.

Note: The more diluted the blood sample, the better the purity of the mononuclear cells. 

2. Pipette 16 mL of Ficoll-Paque" into a Leucosep® Tube. Close the tube and centrifuge at 1000xg for 30 seconds at 20 °C. The Ficoll-Paque is now located below the porous barrier.

3. Transfer diluted cell suspension into the prepared Leucosep Tube and fill up with buffer to a total volume of 50 mL.

4. Centrifuge at 1000xg for 10 minutes at 20 °C in a swinging-bucket rotor without brake. 

5. Three layers occur above the barrier: a plasma layer, the interphase consisting of PBMCs, and a small layer of Ficoll-Paque. Discard the plasma layer.

6. Transfer the interphase above the barrier, which contains the PBMCs, to a new 50 mL conical tube. 

7. Fill the conical tube with buffer, mix, and centrifuge at 300xg for 10 minutes at 20 °C. Carefully remove supernatant completely.

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

9. Repeat step 8. Most of the platelets will remain in the supernatant upon centrifugation at 200xg.

10. Resuspend cell pellet in an appropriate amount of buffer and proceed to magnetic labeling. For details see MACS Cell Separation Reagents data sheets. • Note: PBMCs may be stored in the refrigerator overnight in buffer. 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.

Isolation of Leukocytes for staining

Leukocytes in whole blood are predominately lymphocytes, macrophages/monocytes and granulocytes. If you are interested in staining granulocytes, you need only lyse the RBC. If you are interested in purifying peripheral blood mononuclear cells (lymphocytes, and macrophages/monocytes only), you must purify your PBMC using a density gradient procedure.

Lysing Red blood Cells: Many commercially available reagents are available that lyse RBCs. Most are simply ammonium chloride solutions. RBC lysis buffer can easily and inexpensively be made: 10x RBC Lysis Buffer 90 g. NH4Cl (0.155M) 10 g. KHCO3 (0.01M) 370 mg. EDTA (0.1mM) Dissolve in one liter of ddH2O Filter sterilize through .22 micron filter

This solution can be stored at 4 oC for months. Dilute 1:10 in ddH2O before use. RBC may be lysed before or after staining. Mix 200 µl of whole blood with 2 ml of lysis buffer, incubate at room temperature for 5 minutes, spin down at 300 x g in order to remove lysis buffer. Repeat if necessary. The cells are ready for analysis or staining.

Note: Leukocytes should not be left in lysis buffer for extended periods of time, as cell morphology and viability will be negatively affected