Nov 21, 2020
Version 2
PBMC- 03 - TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit” V.2
- Marco Cosentino1,
- Elisa Storelli1,
- Alessandra Luini1,
- Massimiliano LM 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 LM Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 03 - TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”. protocols.io https://dx.doi.org/10.17504/protocols.io.bpxumpnwVersion 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: 44756
Abstract
List of published work using this procedure:
- Kustrimovic, N., Comi, C., Magistrelli, L., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Minafra, B., Riboldazzi, G., Sturchio, A., Mauri, M., Bono, G., Marino, F., & Cosentino, M. (2018). 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. Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8
Materials
MATERIALS
MACS MultiStandMiltenyi BiotecCatalog #130-042-303
EDTASigma AldrichCatalog #ED2SS
BSASigma AldrichCatalog #A2153
CD4 CD25 Regulatory T Cell Isolation KitMiltenyi BiotecCatalog #130-091-301
LD columnsMiltenyi BiotecCatalog #130-042-901
MS columnsMiltenyi BiotecCatalog #130-042-201
MidiMACS SeparatorMiltenyi BiotecCatalog #130-042-302
MiniMACS SeparatorMiltenyi BiotecCatalog #130-042-102
INSTRUMENTATION REQUIRED
Laminar flow hood (Room PS03)
Before start
Make sure that the buffer is cold (+4°C) by putting it on ice for all the time needed to perform this protocol!
You need to obtaine TEFF and TREG cells uncontaminated for the subsequent cell culture, hence 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).
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).
Determine the cell number and viability with the microscope by staining with either Türk or Trypan blue. You can use also Cellometer machine. (PBMC purity should be ≥95% with few contaminant PMNs to prevent clogging of the column).
For manual cell count use Türk solution for checking purity.
Follow protocol CELL COUNT- 02.
For automatic cell count with Cellometer machine use Trypan Blue for checking viability.
Follow protocol CELL COUNT- 03
Document
NAME
SOLUTION- 08 - Türk solution
CREATED BY
Farmacologia Medica
Document
NAME
SOLUTION- 09 - Trypan Blue solution
CREATED BY
Farmacologia Medica
Equipment
Cellometer Auto T4
NAME
Automated cell counter
TYPE
Nexcelom Bioscience
BRAND
EuroClone
SKU
OPTIONAL STEP
Sorting of TREG is quite long procedure. Especially in clinical studies with whole blood of enrolled subject, it is possible to stop it after PBMC isolation and counting.
In this case, put cells in a flask with SOLUTION- 13 at a concentration of 1x106 cells/mL.
Put the flasks in an incubator (37°C, 5% CO2), and start sorting procedure the day after.
Document
NAME
SOLUTION- 13 - Complete culture medium
CREATED BY
Farmacologia Medica
Centrifuge the obtained PBMCs at 1200 x g, Room temperature, 00:05:00 .
Aspirate supernatant completely. (Use 15 mL-conical tube)
Note
Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
Volumes for magnetic labeling indicated in this procedure are for up to 10x106 total PBMCs. When working with higher than 10x106 cells, scale up all reagent volumes and total volumes accordingly.
For optimal performance it is important to obtain a single-cell suspension before magnetic labeling.
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Resuspend the pellet in 100 µL of cold SOLUTION- 10 (adjust volumes for 10x106 cells).
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Add 10 µL of CD4+T Cell Biotin-Antibody Cocktail (adjust volumes for 10x106 cells).
Mix well and incubate for 00:10:00 at 4 °C
Add 20 µL of Anti-Biotin MicroBeads (adjust volumes for 10x106 cells), mix well and incubate 00:15:00 at 4 °C
Add 5 µL of cold SOLUTION- 10 and centrifuge at 1200 x g, Room temperature, 00:05:00
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Discard the supernatant and resuspend the pellet in 500 µL of cold SOLUTION- 10.
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Place LD column in the magnetic field of suitable MACS Separator (violet, see figures below).
Separator must be attached to the MACS multistand (black) in order to work
Prepare column by rinsing it with 3 mL of cold SOLUTION- 10 (trash the effluent).
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Apply cell suspension onto the column.
Collect unlabeled cells that pass through column. Wait until the column reservoir is completely empty.
Wash again 2 times with 3 mL of cold SOLUTION- 10 and 1 last time with 2 mL of of the same buffer.
Collect total effluent that is consisting of unlabeled pre-enriched CD4+ cell fraction.
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Centrifuge the obtained effluent at 1200 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Remove supernatant and resuspend cell pellet in 100 µL of cold SOLUTION- 10 (adjust the volumes for 10x106 cells).
Note
Use 15 mL-conical tubes.
Volumes for magnetic labeling indicated in this procedure are for an initial starting cell number of up to 10x106 total PBMCs. For higher initial cell numbers, scale up all reagent volumes accordingly.
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Add 10 µL of CD25 MicroBeads (adjust volumes for 10x106 cells), mix well and incubate 00:15:00 at 4 °C in the dark.
Add 5 mL of cold SOLUTION- 10 and centrifuge at 1200 x g, Room temperature, 00:05:00
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Remove the supernatant and resuspend the cell pellet in 500 µL of cold SOLUTION- 10.
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Place the MS column in the magnetic field of a suitable MACS Separator (green, see figures below).
Separator must be attached to the MACS multistand (black) in order to work
Prepare the column by rinsing with 500 µL of cold SOLUTION- 10 and trash the effluent.
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
Apply cell suspension onto the column.
Collect the flow-through containing unlabeled negative fraction (T effector cells CD25-).
Wait until the column reservoir is completely empty, wash again 3 times with 2 µL of cold SOLUTION- 10.
Note
Use 15 mL-conical tube
Remove the column from the magnet and place it on a suitable collection tube.
Note
Use 15 mL-conical tube
Pipette 1 mL of cold SOLUTION- 10 onto the column.
Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
The cells that are flushed out are CD25 labeled cells positive fraction (T regulatory cells CD25+).
Document
NAME
SOLUTION- 10 - TEFF/TREG isolation buffer
CREATED BY
Farmacologia Medica
In order to make sure that collection of cells was complete, repeat the last step TWO more times.
Centrifuge isolated TEFF and TREG at 1200 x g, Room temperature, 00:05:00
Equipment
Allegra AVANTI 30
NAME
Centrifuge
TYPE
Beckman Coulter
BRAND
Beckman Italy
SKU
Resuspend TEFF cells in 1 mL of SOLUTION- 4 and TREG cells in 0.2 mL of SOLUTION- 4.
Count them under microscope or Cellometer machine, according to the appropriate procedure (see step 2 of this protocol).
Document
NAME
SOLUTION- 04 - Wash solution (RPMI/FBS) for PBMC
CREATED BY
Farmacologia Medica
OPTIONAL STEPS
If required, it is possible to check the purity of isolated TEFF and TREG.
Proceed as follows:
- Put PBMCs (0.5x106 cells), Teff (0.5x106 cells) and TREG (at least 0.3x106 cells) into 3 different BD Tubes;
- Centrifuge at 1200 x g, Room temperature, 00:05:00
- Remove the supernatant and resuspend the pellet in 50 µL PBS 1X ;
- Add the adequate antibodies such as: CD4 APC-Cy7 (2.5 µL , BD cat. n. 557871), CD25 PE (10 µL , Miltenyi cat. n. 120-001-311) and CD127 AF647 (10 µL , BD cat. n. 558598) or conjugated to other fluorochromes;
- Incubate for 00:20:00 , in the dark at RT;
- Wash with 1 mL of PBS 1X and centrifuge 1200 x g, Room temperature, 00:05:00 ;
- Resuspend the pellet in 350 µL PBS 1X and leave on ice until FACS acquisition with an appropriate protocol.
Document
NAME
SOLUTION- 02 - Phosphate Buffered Saline (PBS)
CREATED BY
Farmacologia Medica