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.
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 www.invitrogen.com/immunology.
Additional Materials Required
- Mixer allowing both tilting and rotation.
- Magnets: See www.invitrogen.com/magnetselection.
- 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).
- 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.
- 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.
Dynabeads® Washing Procedure
Dynabeads® should be washed before use.
- Resuspend the Dynabeads® in the vial. (i.e. vortex for > 30 sec or tilt and rotate for 5 min.)
- Transfer the desired volume of Dynabeads® to a tube.
- Add the same volume of Isolation Buffer, or at least 1 ml, and mix.
- Place the tube in a magnet for 1 min and discard the supernatant.
- 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 www.invitrogen.com/cellisolation and follow our Quicklinks for recommended sample preparation procedures.
- Resuspend the suspension at 1 x 108 PBMC per ml in Isolation buffer.
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
|Working volume per 5 x 107 PBMC||Working volume per 5 x 108 PBMC|
|Tube size||15 ml||50 ml|
|Cell volume (step 1)||500 μl||5 ml|
|FBS/FCS (step 2)||100 μl||1 ml|
|Antibody Mix (step 3)||100 μl||1 ml|
|Washing (step 5)||10 ml||40 ml|
|Resuspension (step 6)||500 μl||5 ml|
|Depletion MyOne Dynabeads® (step 7)||500 μl||5 ml|
|Volume added before|
magnetic separation (step 10)*
|5 ml||20 ml|
* 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.
- Transfer 500 μl (5 x 107) PBMC in Isolation Buffer to a tube.
- Add 100 μl Blocking Reagent.
- Add 100 μl of Antibody Mix.
- Mix well and incubate for 20 min at 2–8°C.
- 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.
- Resuspend the cells in 500 μl Isolation Buffer.
- Add 500 μl pre-washed Depletion MyOne SA Dynabeads®.
- Incubate for 15 min at 2–8°C with gentle tilting and rotation.
- 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).
- Add 5 ml Isolation Buffer (When working with volume < 1 ml during incubation with beads, add 1 ml Isolation Buffer before resuspension).
- Place the tube in the magnet for 2 min.
- Transfer the supernatant, containing the untouched human monocytes, to a new tube.
- Add 5 ml Isolation Buffer to tube containing the Dynabeads®.
- Resuspend the bead-bound cells by vigorously pipetting as described in step 9.
- Place the tube in the magnet for 2 min.
- Combine the two supernatants.
- Optional: To remove residual beads; place the tube in the magnet for 2 min. and transfer cells to a new tube.
Description of Materials
Depletion MyOne™ SA Dynabeads® are uniform, superparamagnetic polystyrene beads (1.0 μm diameter) coated with streptavidin (SA). The Antibody Mix contains biotinylated mouse IgG antibodies for CD3, CD7, CD16 (specific for CD16a and CD16b), CD19, CD56, CDw123 and CD235a (Glycophorin A). Supplied in PBS with 0.5% BSA and 0.02% sodium azide (NaN3). The Blocking Reagent is aggregated gamma globulin in 0.9% NaCl. The gamma
globulin may precipitate in solution due to high concentration. This is not contamination and the solution can be used after mixing. The Blocking Reagent is added to block the FC- receptors on the monocytes.
This product is stable until the expiry date stated on the label when stored unopened at 2-8°C. Store opened vials at 2-8°C and avoid bacterial contamination. Keep Dynabeads® in liquid suspension during storage and all handling steps, as drying will result in reduced performance. Resuspend well before use.
Warnings And Limitations
This product is for research use only. Not intended for any animal or human therapeutic or diagnostic use unless otherwise stated. Follow appropriate laboratory guidelines. This product contains 0.02% sodium azide as a preservative, which is cytotoxic.
Avoid pipetting by mouth!
Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. When disposing through plumbing drains, flush with large volumes of water to prevent azide build up. Certificate of Analysis (CoA) is available upon request. Material Safety Data Sheet (MSDS) is available at http://www.invitrogen.com.
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