Isolation of Untouched Human Monocytes Cells by Depletion of Non-Monocytes Cells

This protocol is intended for isolation of untouched human monocytes by depletion of non-monocytes (T cells, B cells, NK cells, dendritic cells, erythrocytes, granulocytes and macrophages) from peripheral blood mononuclear cells (PBMC). Isolated monocytes are bead- and antibody-free and are suitable for any downstream application.

Principle of Isolation

Add blocking reagent and a mixture of biotinylated monoclonal antibodies (Antibody Mix) against the non-monocytes to the starting sample. Add Depletion MyOne SA Dynabeads® to bind the non-monocytes during a short incubation. Separate the bead-bound cells with a magnet. Discard the bead-bound cells and use the remaining, untouched human monocytes for any application.


Downstream Applications

Isolated human monocytes can be used in any application, e.g.: Cell culture, generation of monocyte-derived dendritic cells (Mo-DC), functional assays, molecular studies and Flow cytometry. For recommended products and protocols visit Immunology Research Guide. 


Additional Materials Required

• Mixer allowing both tilting and rotation.
• Magnets: See Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps.
• Isolation Buffer: Ca²⁺ and Mg²⁺ free phosphate buffered saline (PBS) (e.g. Gibco cat.no. 10010-023) supplemented with 0.1% BSA and 2mM EDTA.
• Mo-DC Culture Media: RPMI-1640w/10%  Fetal Calf Serum (FCS).
• Heat inactivated Foetal Bovine Serum (FBS)/ Fetal Calf Serum (FCS).
• Lymphoprep® for PBMC preparation (Axis Shield PoC, Norway).

Note:

• BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
• EDTA can be replaced by 0.6% sodium citrate.
• PBS containing Ca²⁺ or Mg²⁺ is not recommended.

Critical notes

• Dynabeads® should be washed before use (see Dynabeads® Washing Procedure).
• Use a mixer that provides tilting and rotation of the tubes to ensure that Dynabeads® do not settle in the tube.
• It is important to pipette roughly the recommended times to prevent trapping of monocytes in the bead-bound cell fraction. Try to avoid air bubbles during pipetting.
• Follow the recommended volumes and incubation times. (Never use less than recommended volume of Dynabeads®)
• It is important to keep cells and buffers cold when working with monocytes.

TOP

Dynabeads® Washing Procedure

Dynabeads® should be washed before use.

1. Resuspend the Dynabeads® in the vial. (i.e. vortex for > 30 sec or tilt and rotate for 5 min.)


2. Transfer the desired volume of Dynabeads® to a tube.


3. Add the same volume of Isolation Buffer, or at least 1 ml, and mix.


4. Place the tube in a magnet for 1 min and discard the supernatant.


5. Remove tube from the magnet and resuspend the washed Dynabeads® in the same volume of Isolation Buffer as the initial volume of Dynabeads® (step 2)

Preparations of PBMC

• Prepare a PBMC suspension according to the recommended sample preparation protocol (low platelet numbers).  Visit Pan Mouse IgG - Depletion or Positive Isolation of Cells from Different Species and follow our Quicklinks for recommended sample preparation procedures.
• Resuspend the suspension at 1 x 10⁸ PBMC per ml in Isolation buffer.

Isolation Procedure

This protocol is based on 5 × 10⁷ PBMC and is scalable from 1 × 10⁷ - 5 × 10⁸ cells according to table 1.

Table 1.  Volumes for human monocyte cell isolation

* Note that the difference in volumes for small vs. large scale protocol is based on getting good working volumes during incubation with beads. A shift from small to large scale parameters may be done at any level of cells processed, as long as the resulting volume during incubation with beads is appropriate for the tubes used (more than 1/10 of total tube volume).

• The starting number of cells for this protocol is 5 x 10⁷  (see preparations above for details)
• Keep temperature low to reduce Monocyte depletion. Pre-cool buffers to 2-8°C.
  

1. Transfer 500 μl (5 x 10⁷) PBMC in Isolation Buffer to a tube.


2. Add 100 μl Blocking Reagent.


3. Add 100 μl of Antibody Mix.


4. Mix well and incubate for 20 min at 2–8°C.


5. Wash the cells by adding 10 ml Isolation Buffer. Mix well by tilting the tube several times and centrifuge at 350 × g for 8 min at 2–8°C. Discard the supernatant.


6. Resuspend the cells in 500 μl Isolation Buffer.


7. Add 500 μl pre-washed Depletion MyOne SA Dynabeads®.


8. Incubate for 15 min at 2–8°C with gentle tilting and rotation.


9. Resuspend the bead-bound cells by vigorously pipetting > 10 times using a pipette with a narrow tip opening, (e.g. a 1000 μl pipette tip or a 5 ml serological pipette).


10. Add 5 ml Isolation Buffer (When working with volume < 1 ml during incubation with beads, add 1 ml Isolation Buffer before resuspension).


11. Place the tube in the magnet for 2 min.


12. Transfer the supernatant, containing the untouched human monocytes, to a new tube.


13. Add 5 ml Isolation Buffer to tube containing the Dynabeads®.


14. Resuspend the bead-bound cells by vigorously pipetting as described in step 9.


15. Place the tube in the magnet for 2 min.


16. Combine the two supernatants.


17. Optional:  To remove residual beads; place the tube in the magnet for 2 min. and transfer cells to a new tube.

1. Andersen MH et al (2005) Spontaneous Immunity against Bcl-xL in Cancer Patients. J. Immunol. 175: 2709-2714.


2. Chemnitz JM et al (2004) SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation but only receptor ligation prevents T cell activation. J. Immunol. 173: 945-954.


3. Jarnak-Jankovic S et al (2005) Evaluation of dendritic cells loaded with apoptotic cancer cells or expressing tumour mRNA as potential cancer vaccines against leukemia. BMC Cancer. 5:20 doi:10.1186/1471-2407-5-20.


4. Monteiro M et al (2007) Cartography of gene expression in CD8 single cells: novel CCR7- subsets suggest differentiation independent of CD45RA expression. Blood. 109:2863-2870.


5. van Rhee F et al (2005) NY-ESO-1 is highly expressed in poor prognosis multiple myeloma and induces spontaneous humoral and cellular immune responses. Blood. (Pre-published online): 10.1182/blood-2004-09-3707.


6. Zioza A et al (2003) CD8+ T cells that express CD4 on their surface (CD4dim-CD8bright T cells) recognize an antigenspecific target, are detected in vivo and can be productively infected by T-tropic HIV. Blood 102: 2156-2164.