Human Samples

Whole Blood and Buffy Coat
Whole blood or buffy coat can be used as starting samples. In some cases, it is necessary to wash the sample to remove interfering soluble factors.

  1. Dilute the whole blood or buffy coat in PBS w/0.1% BSA and 0.6% Na-citrate or 2 mM EDTA (without Ca2+ and Mg2+) (1+2).
  2. Centrifuge at 600 x g for 10 min at 2-8°C.
  3. Discard the plasma fraction/upper layer. Resuspend blood to the original volume in PBS w/0.1% BSA and 0.6% Na-citrate or 2 mM EDTA (without Ca2+ and Mg2+) and buffy coat 1+1 before adding the beads.

MNC

  1. Collect sample with anticoagulant present (EDTA, ACD, heparin). Dilute peripheral blood 1 + 1, buffy coat 1 + 2, bone marrow 1+1 and umbilical cord blood 1 + 3 in PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA.
  2. Layer up to 35 ml of the diluted sample over 15 ml gradient medium in a 50 ml tube.
  3. Centrifuge for 800 x g for 20 minutes at 18-25°C. If blood is stored for more than 2 hours, increase centrifugation time to 30 min (as per gradient medium instructions).
  4. Collect MNC from the interface and transfer cells to a 50 ml tube.
  5. Wash MNC three times with PBS w/0.1% BSA by centrifugation at 300 x g for 8 min at 2-8°C.
  6. Resuspend the cells to 1 x 107 per ml in PBS w/0.1% BSA and cool to 2-8°C

Preparation of MNC from Buffy Coat to Obtain Low Platelet Numbers

  1. Dilute 10 - 18 ml buffy coat with PBS w/ 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18-25°C.
  2. Add the diluted buffy coat on top of 15 ml of Lymphoprep™.
  3. Centrifuge at 160 x g for 20 min at 20°C. Allow to decelerate without brakes.
  4. Remove 20 ml of supernatant to eliminate platelets.
  5. Centrifuge at 350 x g for 20 min at 20°C. Allow to decelerate without brakes.
  6. Recover MNC from the plasma/Lymphoprep interface and transfer the cells to a 50 ml tube.
  7. Wash MNC once with PBS w/ 0.1% BSA by centrifugation at 400 x g for 8 min at 2-8°C.
  8. Wash MNC twice with PBS w/ 0.1% BSA by centrifugation at 225 x g for 8 min at 2-8°C and resuspend the MNC at 1 x 108 MNC per ml in PBS w/ 0.1% BSA.

Tissue Digests
Follow standard tissue preparations using enzymes and mechanical disruption to get a single cell suspension. Eliminate large aggregates by sieving the digested cell suspension through a cell strainer. Disruption of tissue normally results in some cell death and release of DNA. Free DNA will impair cell capture, recovery and purity. DNase I treatment is performed by incubating the cell suspension in PBS w/ 0.1% BSA + 1mM CaCl2 + 0.5 mM MgCl2 at 18-25°C for 30 min with 120 Kunitz units DNase I per ml.  (For CELLection products, wash cells to remove DNase before adding the beads.)

Bone Marrow (CD34)
Washing and DNase treatment is recommended.

  1. Mix 2 ml (107-108 cells) bone marrow with 2 ml PBS w/ 0.1% BSA + 0.6% Na-citrate.
  2. Centrifuge at 600 g for 8 min at 18-25 °C.
  3. Discard the supernatant and resuspend to 5 ml with PBS w/ 0.1% BSA + 1mM CaCl2 + 0.5 mM MgCl2.
  4. Add 600 Kunitz units DNase I (120 Kunitz units DNase I per ml).
  5. Incubate cells for 30 minutes at 18-25 °C with both gentle tilting and rotation.
  6. Centrifuge cell suspension for 8 minutes, 600 x g, at 18-25 °C.
  7. Discard supernatant and resuspend cell pellet in PBS w/ 0.1% BSA.
  8. Repeat steps 6-7 once.
  9. Resuspend at 1 x 108 cells per ml in RPMI 1640 / 1% FCS

Mouse Samples

Tissue Digests
Mouse cells are generally harvested from spleen, thymus or lymph node.
Cells may be squeezed from a whole organ, dissociated by enzyme digestion and teased out by fine chopping followed by sieving through a cell strainer, etc. Persistent clumps can be removed by sedimentation. If necessary, red cells can be removed by lysing.

Spleen and Lymph Node Cells
Steps 1-2 and 4-6 are performed at 18-25°C, the other steps are performed with cold media or buffers at 2-8°C.
Use a recently killed mouse. Remove the spleen or the lymph nodes from the mouse and transfer the tissues to a tube containing 30 ml of PBS w/ 0.1% BSA + 0.6% Na-citrate (without Ca2+ and Mg2+) or 2 mM EDTA preheated to 18-25°C.

  1. Using a pastette, transfer the tissue to a cell strainer on top of a 50 ml tube. Use the back of a syringe plunger to macerate the cells through the filter. Rinse the filter at regular intervals with PBS w/ 0.1% BSA + 0.6% Na-citrate (without Ca2+ and Mg2+) .
  2. Fill the tube with cold PBS w/ 0.1% BSA + 0.6% Na-citrate (without Ca2+ and Mg2+) and centrifuge at 300 x g at 2-8°C for 10 min.
  3. Discard the supernatant and resuspend the cells in 50 ml cold PBS w/ 0.1% BSA + 0.6% Na-citrate (without Ca2+ and Mg2+). Centrifuge at 300 x g at 2-8°C for 10 min.
  4. Discard the supernatant and resuspend the cells in 5-10 ml PBS w/ 0.1% BSA with Ca2+ and Mg2+ at 18-25 °C.
  5. Add 120 Kunitz units DNase per ml of cell suspension. Mix gently, incubate on a mixer for 15 min at 18-25 °C. Mix vigorously.
  6. Filter through a cell strainer.
  7. Count the leucocytes (lyse the red blood cells for accurate cell counting).
  8. Fill the tube with cold PBS w/ 0.1% BSA + 0.6% Na-citrate (without Ca2+ and Mg2+) and centrifuge at 300 x g at 2-8°C for 10 min.
  9. Discard the supernatant and resuspend the cells in PBS w/ 0.1% BSA to 1 x 107 cells/ml.

Lysis of Mouse Erythrocytes (Red Cell Lysis):

  1. Harvest mouse spleen and prepare a single cell suspension.
  2. Pellet the cells by centrifugation (300-400xg) at 2-8°C and aspirate the supernatant.
  3. Resuspend the pellet in 5 ml/spleen of Lysis Buffer.
  4. Incubate at room temperature (or on ice, if preferred) for 4-5 minutes with occasional shaking.
  5. Stop the reaction by diluting the Lysis Buffer with 20-30 ml of 1X PBS.
  6. Spin the cells (300-400xg) at 2-8°C, carefully remove the supernatant and resuspend the pellet in the appropriate buffer for use in the next step of your experimental procedure.
  7. Count the cells.

Lysis buffer is formulated for optimal lysis of erythrocytes in single-cell suspensions of mouse hematopoietic tissues such as spleen. Lysis buffer contains ammonium chloride, which lyses red cells with minimal effect on lymphocytes when used as instructed. Nucleated red cells are not effectively lysed with ammonium chloride. RBC lysis is not necessary when working with mouse thymus and lymph nodes.
(eBioscience: 1X RBC Lysis Buffer. Catalog number 00-4333-57)
(BioLegend: Lysis Buffer (10X).Catalog Number: 420301)

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