This product is intended for positive magnetic isolation of CD4+ T cells ffrom human peripheral blood mononuclear cells (PBMC's). In the first step. FlowComp Human CD4 Antibody (mouse IgG1 antibody against human CD4) is added and will bind to the target cells. In the second step, CD4+ T cells, that have bound the specific antibodies are captured by the Dynabeads. In the third and last step, the cells are released by the Dynabeads.
Isolated cells are bead-free and may be used directly in any downstream application including flow cytometry. The cells readily proliferate in response to Dynabeads ® Human T-Activator CD3/CD28 (Cat. no.111.31D/111.32D/61D) and can be measured by incorporation of EdU (e.g. Click-IT™-EdU Cat. no A10202) or in a CFSE assay (Invitrogen Cat. no. C34554). For recommended products and protocols visit: the Immunology Research Guide.
- Isolation Buffer: Ca2+ and Mg2+ free phosphate buffered saline (PBS) (e.g. Invitrogen Gibco Cat. no.14190) supplemented with 0.1% BSA and 2mM EDTA. BSA can be replaced by human serum albumin (HSA) or 2% FBS/FCS.
- Mixer allowing both tilting and rotation.
- Dynal magnet: see Dynabeads® mRNA Purification Kit for mRNA Purification from Total RNA preps for magnet recommendations.
- Optional: Flow cytometry antibody reagents. Invitrogen recommends using anti-CD3-Alexa Fluor 488 (Invitrogen Cat. no. MHCD0320), and anti-CD4-PE (Invitrogen Cat. no. MHCD0404) as primary fluorescent antibodies for flow staining of cells after isolation. See the Immunology Research Guide for recommended products and protocols.
- Optional: For viability analysis, SYTOX® Red (Invitrogen Cat. no. S34859) is recommended.
- Use a mixer that provides tilting and rotation of the tubes to ensure that the Dynabeads do not settle in the tube.
- Resuspend the Dynabeads in the vial carefully before use, i.e. vortex for > 30 sec, or tilt and rotate for 5 minutes.
- This product should not be used with Dynal® MPC™ -1 (Invitrogen mDynal Cat. no. 120.01D).
- Avoid air bubbles during pipetting.
- Never use less than recommended pipetting volumes and incubation times.
- Keep all buffers cold.
- Approximately 1 x 106 T cells are present per ml human blood, and about 70% of these T cells strongly express the CD4 antigen.
- This protocol describes magnetic capture and isolation of CD4+ T cells without contamination of CD4 expressing monocytes, starting from 5 x 107 PMBCs and using Dynabeads® FlowComp™ Human CD4. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.
- Isolate PBMCs from anti-coagulayted peripheral blood or leukocyte enriched buffy coat using standard procedure. (see section Technical Advise for further information).
- Prepare a single cell suspension of 1 x 108 cells/ml in Isolation Buffer.
- Prepare approximately 10 ml. of Isolation Buffer per 5 x 107 cells.
All incubations at room temperature (RT) can also be performed at 2-8° C
- Use 500 μl (5 x 107 cells) form the preparation above, resuspend and add 25 μl FlowComp Human CD4 Antibody.
- Mix well and incubate for 10 minutes at 2-8° C.
- Add 2 ml. Isolation Buffer to wash cells, followed by centrifugation for 8 minutes at 350 x g.
- Remove and discard the supernatant.
- Add 1 ml Isolation Buffer to the cell pellet and resuspend.
- Add 75 μl resuspended FlowComp Dynabeads and mix well.
- Incubate for 15 min at RT under rolling and tilting
- Place the tube on the magnet for a minimum 1 minute. Carefully remove and discard the supernatant.
- Remove the tube from the magnet. Add at least 1 ml. Isolation Buffer and resuspend the bead-bound cells by gentle pipetting 5 times.
- Place the tube on the magnet for minimum 1 minute. Carefully remove and discard the supernatant.
- Remove the tube from the magnet and carefully resuspend the bead-bound cells in 1 ml Flowcomp Release Buffer.
- Incubate for 15 min at RT under rolling and tilting
- Mix the cells by pipetting 10 times and place the tube in the magnet for 1 minute.
- Transfer the supernatant containing the bead-free cells to a new tube and again place on the magnet for 1 minute to remove and residual beads.
- Transfer supernatant containing the beads-free cells to a new tube.
- Add 2 ml Isolation Buffer followed by centrifugation for 8 minutes at 350 x g.
- Discard the supernatant and resuspend the cell pellet in preferred medium.
- Keep the cells on 2-8° C until further use in downstream applications.
- To avoid unspecific labeling of cells during flow staining, we recommend to use gammaglobulin prior to staining with primary fluorescent antibody.
- For better purity, repeat the washing step once or transfer the bead-bound cells to a new tube before adding the FlowComp™ Release Buffer
Preparation of PBMC from Buffy Coat
This method gives low platelet numbers and is recommended for use with Dynabeads FlowComp™ products.
- Dilute 10–18 ml buffy coat with PBS with 0.1% BSA + 0.6% Na-citrate or 2 mM EDTA to a total volume of 35 ml at 18–25°C.
- Add the diluted buffy coat on top of 15 ml of Lymphoprep™.
- Centrifuge at 160 × g for 20 minutes at 20°C. Allow to decelerate without brakes.
- Remove 20 ml of supernatant to eliminate platelets.
- Centrifuge at 350 × g for 20 minutes at 20°C. Allow to decelerate without brakes.
- Recover PBMC from the plasma/Lymphoprep ™ interface and transfer the cells to a 50 ml tube.
- Wash PBMC once with PBS with 0.1% BSA by centrifugation at 400 × g for 8 minutes at 2–8°C.
- Again wash PBMC with PBS with 0.1% BSA by centrifugation at 225 × g for 8 minutes at 2–8°C and resuspend the cells at 1 × 108 PBMC per ml in PBS w/0.1% BSA.
For starting samples other than buffy coat, please see www.invitrogen.com/cellisolation for recommended PMBC preparation procedures. The latest revision of the package insert/instruction for use is available on this website.