Nov 21, 2020
Version 2
PBMC- 02 - CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit” V.2
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano Legnaro1,
- Emanuela Rasini1,
- Marco Ferrari1,
- Franca Marino1
- 1Center for Research in Medical Pharmacology, University of Insubria (Varese, Italy)
Protocol Citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 02 - CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit”. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxqmpmwVersion created by Farmacologia Medica
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: November 21, 2020
Last Modified: November 21, 2020
Protocol Integer ID: 44752
Keywords: dopaminergic receptors on cd4, dynabeads cd4 positive isolation kit, patients with parkinson, parkinson, treg in drug, cd4 positive isolation kit, dopaminergic receptor, dopamine receptor gene polymorphism, functional th1 bias, influence of dopamine receptor gene polymorphism, cd4, pbmc, journal of neuroinflammation
Abstract
List of published works using this protocol:
- Kustrimovic N., Comi C., Magistrelli L., Rasini E., Legnaro M., Bombelli R., Aleksic I., Blandini F., Minafra B., Riboldazzi G., Struchio A., Mauri M., Bono G., Marino F., Cosentino M. Parkinson’s disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients (2018). Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8
- Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738
- Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032
Materials
MATERIALS
Dynabeads™ CD4 Positive Isolation KitThermo FisherCatalog #11331D
Fetal Bovine Serum (FBS)EuroCloneCatalog #ECS0180L-500 ml
RPMI 1640EuroCloneCatalog #ECM 0495L- 500 ml
BSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A2153
BD tubes Becton Dickinson (BD)Catalog #352054
Instrumentation required:
a.Magnet (DynaMag™)
b.Sample Mixer with rotation
c.Laminar flow hood
Troubleshooting
Before start
If you need to obtain CD4+ T cell for subsequent cell culture, make sure you are using sterile buffers and sterile plastic disposables as well. Moreover, work under laminar flow hood when you are processing samples (from the beginning to the end of the following procedure). Otherwise, use non-sterile Buffers and disposables, and process samples in a cell isolation laboratory.
IMPORTANT NOTE: the isolation protocol is calibrated for using 25µL of beads for 10x106 PBMCs resuspended in 1mL. For lower or higher cell number than 10x106, resize the volumes, accordingly. (See also Table 1on the data sheet of the kit).
ALL REAGENTS MUST BE AT ROOM TEMPERATURE WHEN USED!!!
Isolate PBMCs according either to the standard protocol from fresh blood or from buffy coat (PBMC- 01a - Isolation of Human PBMC from Buffy Coat, PBMC- 01b - Isolation of Human PBMC from Whole Blood).
Count the cells with Cellometer machine or by manual count, using either Trypan Blue or Türk solutions accordingly.
For automatic cell count with Cellometer machine use Trypan Blue.
Follow protocol CELL COUNT- 03
For manual cell count use Türk solution for checking purity.
Follow protocol CELL COUNT- 02
Equipment
Cellometer Auto T4
NAME
Automated cell counter
TYPE
Nexcelom Bioscience
BRAND
EuroClone
SKU
Resuspend Dynabeads in the vial using a vortex for >30 sec.
Transfer the desired volume of Dynabeads to a 5mL-tube (use BD tubes cat. n. 352054) following this proportion: 25µL of beads for 10x106cells.
Add 2 µL of Solution- 11 (found in the kit materials as Buffer 1), resuspend and place the tube into the magnet: beads will attach to the magnet very quickly (few seconds).
Discard then the supernatant by using a glass Pasteur pipette.
Remove the tube from the magnet.
Repeat the washing step 2 or 3 times to make sure that DMSO is all washed up.
After counting, centrifuge PBMCs sample at 1200 x g, 00:05:00 .
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Discard supernatant and resuspend pellet of 10x106 cells in 1 mL of SOLUTION- 11.
Transfer cell suspension into the tube with beads, and resuspend vigorously.
Incubate the beads with cells for 00:20:00 at 4 °C with gentle rotation by putting the Sample Mixer in the fridge.
After incubation place the tube on the magnet and wait for 1-2 minutes, that is until the complex beads-cells is completely attached to the magnet.
While the tube is still in the magnet, carefully remove and discard the supernatant with a glass Pasteur pipette.
Remove the tube from the magnet, add 2 µL of SOLUTION- 11 and resuspend the cells very vigorously because of aggregates.
Repeat steps 11-13 twice (in total 3 times) to wash the bead-bound CD4+ T cells. These steps are critical to obtain a high purity of isolated cells.
Resuspend cell pellet in 100 µL of SOLUTION- 07 (found in the kit materials as Buffer 2)
[The volume is calibrated for 10x106 cells, for lower or higher number of cell resize the volume accordingly].
Add 10 µL of DETACHaBEAD® CD4 for each 10x106 PBMCs.
(Resize this volume if the number of starting cell is different)
Add another 500 µL of SOLUTION- 07 to increase the volume and transfer everything in a 1.5 mL eppendorf.
Incubate 00:45:00 at Room temperature (RT) with gentle rotation by using a Sample Mixer.
Transfer the sample from eppendorf to BD tube, and place the tube on magnetand wait for 1-2 mins, that is until the complex beads-cells is completely attached to the magnet.
While the tube is still in the magnet, transfer the supernatant containing the released cells into a 15 mL conical tube.
To obtain residual cells, wash the beads 3 times with 500 µL of SOLUTION- 07 and collect the supernatant each time.
Add to the detached cell suspension SOLUTION- 07 to a final volume of 5 mL and centrifuge at 1200 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Resuspend the cells for cell counting in 1 mL of SOLUTION- 07: follow the appropriate protocol (see step 2 of this protocol).
Check the viability with Trypan blue.
OPTIONAL STEP
Check the purity of the isolated CD4+ T cells by flow cytometry.
If needed, check the purity by labeling CD4 with the appropriate CD markers, such as CD3, CD4, CD8 and CD14 Ab and analyze samples with a flow cytometer to exclude the presence of undesired subsets.
Equipment
BD FACS Celesta
NAME
Flow Cytometer
TYPE
Becton Dickinson
BRAND
Milan Italy BD
SKU
EXPECTED RESULTS
Expected result
Cell Viability: ≥95 %
Cell Yield: ± 4,6 x106 cells starting from 25 mL of Fresh Blood
± 6 x106 cells starting from 25 mL of Buffy Coat
If checked, purity of the isolated CD4+ cells must be ≥95 %