Nov 02, 2023

Public workspaceTwo-step method for isolation of inactivated CD4+ T-cells from human blood mononuclear cells

DOI
dx.doi.org/10.17504/protocols.io.36wgq3mxolk5/v1
  • 1Central Research Institute of Epidemiology
  • Anna Esman: https://www.researchgate.net/profile/Anna-Esman
  • Michael Vinokurov: https://www.researchgate.net/profile/Michael-Vinokurov-2
Anna Esman
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DOI: dx.doi.org/10.17504/protocols.io.36wgq3mxolk5/v1
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 Acknowledgements:
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. ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D

2. ReagentCELLection™ Biotin Binder KitThermofisherCatalog #11533D
3. ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
4. ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
5. ReagentHLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
6. ReagentCD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82

and magnetic tube separator.
Materials
1. ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D

2. ReagentCELLection™ Biotin Binder KitThermofisherCatalog #11533D
3. ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
4. ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
5. ReagentHLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
6. ReagentCD69 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) ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
10. 0.1% BSA
11. Concentration2 millimolar (mM) EDTA
12. ReagentFetal Bovine SerumGibco - Thermo FischerCatalog #26140079
13. ReagentRPMI-1640Pan-EcoCatalog #C310p




Protocol materials
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
ReagentCD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentFetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #26140079
ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
ReagentCELLection™ Biotin Binder KitThermofisherCatalog #11533D
ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
ReagentRPMI-1640Pan-EcoCatalog #C310p
ReagentHLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
ReagentHLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
ReagentCD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
ReagentBD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinBD BiosciencesCatalog #362753
ReagentPBS, pH 7.4Thermo FisherCatalog #10010023
ReagentHLA-DR Monoclonal Antibody (LN3), BiotineBioscienceCatalog #13-9956-82
ReagentCD69 Monoclonal Antibody (FN50), BiotineBioscienceCatalog #13-0699-82
ReagentDynabeads™ Untouched™ Human CD4 T Cells KitThermofisherCatalog #11346D
ReagentCELLection™ Biotin Binder KitThermofisherCatalog #11533D
ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))eBioscienceCatalog #14-0719-82
ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBioscienceeBioscienceCatalog #13-0259-82
ReagentBD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinBD BiosciencesCatalog #362753
Before start
SampleSample
ReagentBD 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 Amount5 mL of human whole blood to the ReagentBD Vacutainer® CPT™ Mononuclear Cell Preparation Tube - Sodium HeparinPan-EcoCatalog #362753

Store tube upright at TemperatureRoom 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.

Centrifigation1500-1800 rcf, 18-25°C in a horizontal rotor (swing-out head) for a minimum of Duration00:15:00
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 Amount15 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
ReagentPBS, pH 7.4Pan-EcoCatalog #10010023 supplemented with 0.1% BSA and Concentration2 millimolar (mM) EDTA.


Preparation of magnetic particles
Resuspend theReagentDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D in the vial (i.e. vortex for > Duration00:00:30 or tilt and rotate for Duration00:05:00 )

5m 30s
Transfer the desired volume ofReagentDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D to a tube.
Add the same volume of Isolation Buffer, or at least Amount1 mL and resuspend.
Place the tube in a magnet for Duration00:01:00 and discard the supernatant.

1m
Remove the tube from the magnet and resuspend the washedReagentDynabeads™ Untouched™ Human CD4 T Cells KitPan-EcoCatalog #11346D in the same volume of Isolation Buffer as the initial volume of Dynabeads®.
Isolation Procedure
Transfer Amount100 µL (5 × 107) PBMC in Isolation Buffer to a tube.

Add Amount20 µL ReagentPBS, pH 7.4Pan-EcoCatalog #10010023

Add Amount20 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 Duration00:20:00 at Temperature2-8 °C

20m
Wash the cells by adding Amount2 mL Isolation Buffer. Mix well by tilting the
tube several times and Centrifigation350 x g, 2-8°C, 00:08:00 . Discard the supernatant.
8m
Resuspend the cells in Amount100 µL Isolation Buffer.

Add Amount100 µL pre-washed Dynabeads‱.
Incubate for Duration00:15:00 at TemperatureRoom temperature with gentle tilting and rotation

15m
Add Amount1 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 Duration00:02:00 . Transfer the supernatant containing the untouched human CD4+ T cells, to a new larger tube.

2m
Add Amount2 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 Duration00:02:00 and then combine the two supernatants.
Note
To remove residual beads; place the tube in the magnet for Duration00:02:00 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
46m 30s
Preparing buffers for operations
• Buffer 1: ReagentPBS, pH 7.4Pan-EcoCatalog #10010023 supplemented with 0.1% bovine serum albumin (BSA), Ph7.4 • Buffer 2: ReagentPBS, pH 7.4Pan-EcoCatalog #10010023 with 0.1% BSA and 0.6% sodium citrate or Concentration2 millimolar (mM) EDTA.

Prepare Release Buffer
1. For each vial of freeze-dried DNase I, transfer Amount300 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 Temperature-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 >Duration00:00:30 or tilt and rotate for Duration00:05:00 . Transfer the desired volume of Dynabeads® to a tube (Amount25 µL for one sample).

5m 30s
Add the same volume of Buffer 1, or at least Amount1 mL and resuspend.

Place the tube in a magnet for Duration00:01:00 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 ~Amount10 µg primary biotinylated antibody to Amount1 mL cell suspension and mix ReagentCD71 (Transferrin Receptor) Monoclonal Antibody (OKT9 (OKT-9))Pan-EcoCatalog #14-0719-82 ReagentCD25 Monoclonal Antibody (BC96), Biotin, eBiosciencePan-EcoCatalog #13-0259-82 ReagentHLA-DR Monoclonal Antibody (LN3), BiotinPan-EcoCatalog #13-9956-82 ReagentCD69 Monoclonal Antibody (FN50), BiotinPan-EcoCatalog #13-0699-82

Incubate for Duration00:10:00 at Temperature2-8 °C
10m
Wash the cells by adding Amount2 mL Buffer 2 and Centrifigation350 x g, 00:08:00 . Discard the supernatant.
8m
Suspend the cells in Amount4 mL Buffer 2.
Add Amount25 µL pre-washed and resuspended Dynabeads‱
Incubate for Duration00:20:00 at Temperature2-8 °C with gentle tilting and rotation.
20m
Place the tube in the magnet for Duration00:02:00 . Transfer the supernatant containing the inactivated human CD4+ T-cells to a new larger tube.
2m
Add Amount4 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