Nov 02, 2023
Two-step method for isolation of inactivated CD4+ T-cells from human blood mononuclear cells
- 1Central Research Institute of Epidemiology
- Anna Esman: https://www.researchgate.net/profile/Anna-Esman
- Michael Vinokurov: https://www.researchgate.net/profile/Michael-Vinokurov-2
Protocol Citation: Anna Esman, Michael Vinokurov 2023. Two-step method for isolation of inactivated CD4+ T-cells from human blood mononuclear cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3mxolk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 24, 2023
Last Modified: November 02, 2023
Protocol Integer ID: 89818
Keywords: CD4+ cells , magnetic separation , untouched human CD4+ T cells, CD4+ inactivated cells, CD4+ T-cells
Funders Acknowledgement:
Effect of DNA methylation in HIV-1 positive individuals on viral reservoir reactivation
Grant ID: 122053000056-2
Abstract
1. Obtaining human CD4+ T cells
2. Obtaining CD4+ inactivated cells
using
1. Dynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
2. CELLection™ Biotin Binder KitThermofisherCatalog #11533D
3. CD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
4. CD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
5. HLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
6. CD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
and magnetic tube separator.
Materials
1. Dynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
2. CELLection™ Biotin Binder KitThermofisherCatalog #11533D
3. CD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
4. CD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
5. HLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
6. CD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
7. Magnetic separator for 1.5 / 5 / 15 / 50 ml tubes
8. Mixer with tilt and rotation capabilities
9. PBS (Ca 2+ and Mg 2+ free) PBS, pH 7.4Thermo FisherCatalog #10010023
10. 0.1% BSA
11. 2 millimolar (mM) EDTA
12. Fetal Bovine SerumGibco - Thermo FischerCatalog #26140079
13. RPMI-1640Pan-EcoCatalog #C310p
Protocol materials
Dynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
In Materials, Abstract and 3 steps
Fetal Bovine SerumGibco - Thermo FischerCatalog #26140079
Materials
CELLection™ Biotin Binder KitThermofisherCatalog #11533D
In Materials, Abstract
RPMI-1640Pan-EcoCatalog #C310p
Materials
HLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
In Materials, Abstract, Step 8.1
BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinBD BiosciencesCatalog #362753
Before starting, Step 1.1
PBS, pH 7.4Thermo FisherCatalog #10010023
In Materials and 4 steps
CD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
In Materials, Abstract, Step 8.1
CD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
In Materials, Abstract, Step 8.1
CD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
In Materials, Abstract, Step 8.1
Before start
Sample
BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinBD BiosciencesCatalog #362753
Negative depletion of CD4+ T-cells
Negative depletion of CD4+ T-cells
Preparation of PBMC (peripheral blood mononuclear cells)
Collect at least 5 mL of human whole blood to the BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinPan-EcoCatalog #362753
Store tube upright at Room temperature until centrifugation.
Note
To ensure proper barrier formation, blood samples should be centrifuged
within 2 hours of blood collection. Centrifugation more than 2 hours after
specimen collection may cause incomplete barrier formation.
1500-1800 rcf, 18-25°C in a horizontal rotor (swing-out head) for a minimum of 00:15:00
15m
Aspirate approximately half of the plasma without disturbing the cell layer.
Note
Mononuclear cells and platelets will be in a whitish layer just under the plasma layer
Collect cell layer with a Pasteur Pipette and transfer to a 15 mL size conical centrifuge tube
with cap.
Note
Collection of cells immediately following centrifugation will yield best results
Note
An alternative procedure for recovering the separated mononuclear
cells is to resuspend the cells into the plasma by inverting the unopened
BD Vacutainer‱ CPT‱ Tube gently 5 to 10 times.
This is the preferred method for storing or transporting the separated sample for up to 24 hours after centrifugation. To collect the cells, open the BD Vacutainer‱ CPT‱ Tube and
pipette the entire contents of the tube above the gel into a separate vessel.
Expected result
Separation of PBMC from whole blood.
Preparation of the Isolation buffer
PBS, pH 7.4Pan-EcoCatalog #10010023 supplemented with 0.1% BSA and 2 millimolar (mM) EDTA.
Preparation of magnetic particles
Resuspend theDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D in the vial (i.e. vortex for > 00:00:30 or tilt and rotate for 00:05:00 )
5m 30s
Transfer the desired volume ofDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D to a tube.
Add the same volume of Isolation Buffer, or at least 1 mL and resuspend.
Place the tube in a magnet for 00:01:00 and discard the supernatant.
1m
Remove the tube from the magnet and resuspend the washedDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D in the same volume of Isolation Buffer as the initial volume of Dynabeads®.
Isolation Procedure
Transfer 100 µL (5 × 107) PBMC in Isolation Buffer to a tube.
Add 20 µL PBS, pH 7.4Pan-EcoCatalog #10010023
Add 20 µL of Antibody Mix
Note
Contains mouse IgG antibodies towards human CD8, CD14, CD16 (specific for CD16a and
CD16b), CD19, CD36, CD56, CDw123 and CD235a (Glycophorin A)
Mix well and incubate for 00:20:00 at 2-8 °C
20m
Wash the cells by adding 2 mL Isolation Buffer. Mix well by tilting the
tube several times and 350 x g, 2-8°C, 00:08:00 . Discard the supernatant.
8m
Resuspend the cells in 100 µL Isolation Buffer.
Add 100 µL pre-washed Dynabeads‱.
Incubate for 00:15:00 at Room temperature with gentle tilting and rotation
15m
Add 1 mL Isolation Buffer.
Note
When working with lower cell volumes, never use less than 1 mL Isolation Buffer
Resuspend the bead-bound cells thoroughly by pipetting >10 times using a pipette with a narrow tip opening. Avoid foaming.
Place the tube in the magnet for 00:02:00 . Transfer the supernatant containing the untouched human CD4+ T cells, to a new larger tube.
2m
Add 2 mL Isolation Buffer to the tube containing the Dynabeads‱ and resuspend the bead-bound cells by pipetting as described in step 4.10.
Place the tube in the magnet for 00:02:00 and then combine the two supernatants.
Note
To remove residual beads; place the tube in the magnet for 00:02:00 and transfer cells to a new tube.
Expected result
The supernatant contains negatively isolated human CD4+ T-cells.
2m
Negative depletion of inactivated CD4+ T-cells
Negative depletion of inactivated CD4+ T-cells
46m 30s
Preparing buffers for operations
• Buffer 1: PBS, pH 7.4Pan-EcoCatalog #10010023 supplemented with 0.1% bovine serum albumin (BSA), 7.4
• Buffer 2: PBS, pH 7.4Pan-EcoCatalog #10010023 with 0.1% BSA and 0.6% sodium citrate or 2 millimolar (mM) EDTA.
Prepare Release Buffer
1. For each vial of freeze-dried DNase I, transfer 300 µL from the Releasing Buffer Component
II to each tube of Releasing Buffer Component I (DNase I).
Note
Dissolve the enzyme gently. Vigorous mixing will destroy the enzyme.
2. Aliquot the reconstituted Release Buffer into suitable portions. Store at -20 °C . Thaw immediately before use and keep on ice once thawed. Thawed Release Buffer can be re-frozen once.
Wash Dynabeads‱
Resuspend the Dynabeads® in the vial (i.e. vortex for >00:00:30 or tilt and rotate for 00:05:00 . Transfer the desired volume of Dynabeads® to a tube (25 µL for one sample).
5m 30s
Add the same volume of Buffer 1, or at least 1 mL and resuspend.
Place the tube in a magnet for 00:01:00 and discard the supernatant.
1m
Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.
Isolate Cells (Labeling Cells with Biotinylated Antibodies)
Add ~10 µg primary biotinylated antibody to 1 mL cell suspension and mix CD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))Pan-EcoCatalog #14-0719-82 CD25 Monoclonal Antibody (BC96), Biotin, eBiosciencePan-EcoCatalog #13-0259-82 HLA-DR Monoclonal Antibody (LN3), BiotinPan-EcoCatalog #13-9956-82 CD69 Monoclonal Antibody (FN50), BiotinPan-EcoCatalog #13-0699-82
Incubate for 00:10:00 at 2-8 °C
10m
Wash the cells by adding 2 mL Buffer 2 and 350 x g, 00:08:00 . Discard the supernatant.
8m
Suspend the cells in 4 mL Buffer 2.
Add 25 µL pre-washed and resuspended Dynabeads‱
Incubate for 00:20:00 at 2-8 °C with gentle tilting and rotation.
20m
Place the tube in the magnet for 00:02:00 . Transfer the supernatant containing the inactivated human CD4+ T-cells to a new larger tube.
2m
Add 4 mL Buffer 2 to the tube containing the Dynabeads‱ and repeat step 8.7
Combine the two supernatants.
Expected result
The resulting supernatant contains the inactivated human CD4+ T cells.
Protocol references