TITLE: PBMC- 03 - TEFF+TREG Isolation from PBMC with “Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit”
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">List of published work using this procedure:</div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. 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
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?. OPTIONAL STEPSorting 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.
?. Centrifuge the obtained PBMCs at  . Aspirate supernatant completely. (Use 15 mL-conical tube)
Centrifuge: 1200 34, 5 min
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.
?. Resuspend the pellet in  of cold SOLUTION- 10 (adjust volumes for 10x106 cells).
100 µl
?. Add  of CD4+T Cell Biotin-Antibody Cocktail (adjust volumes for 10x106 cells).
10 µl
?. Mix well and incubate for  at
4 °C
?. Add  of Anti-Biotin MicroBeads (adjust volumes for 10x106 cells), mix well and incubate  at
20 µl
4 °C
?. Add  of cold SOLUTION- 10 and centrifuge at
5 µl
Centrifuge: 1200 34, 5 min
?. Discard the supernatant and resuspend the pellet in  of cold SOLUTION- 10.
500 µl
?. Place LD column in the magnetic field of suitable MACS Separator (violet, see figures below).
?. Prepare column by rinsing it with  of cold SOLUTION- 10 (trash the effluent).
3 mL
?. 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  of cold SOLUTION- 10 and 1 last time with  of of the same buffer. Collect total effluent that is consisting of unlabeled pre-enriched CD4+ cell fraction.
3 mL
2 mL
?. Centrifuge the obtained effluent at
Centrifuge: 1200 34, 5 min
?. Remove supernatant and resuspend cell pellet in  of cold SOLUTION- 10 (adjust the volumes for 10x106 cells).
100 µl
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.
?. Add of CD25 MicroBeads (adjust volumes for 10x106 cells), mix well and incubate  at  in the dark.
10 µl
4 °C
?. Add  of cold SOLUTION- 10 and centrifuge at
5 mL
Centrifuge: 1200 34, 5 min
?. Remove the supernatant and resuspend the cell pellet in  of cold SOLUTION- 10.
500 µl
?. Place the MS column in the magnetic field of a suitable MACS Separator (green, see figures below).
?. Prepare the column by rinsing with  of cold SOLUTION- 10 and trash the effluent.
500 µl
?. 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 of cold SOLUTION- 10.
2 µl
Use 15 mL-conical tube
?. Remove the column from the magnet and place it on a suitable collection tube.
Use 15 mL-conical tube
?. Pipette 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+).
1 mL
?. In order to make sure that collection of cells was complete, repeat the last step TWO more times.
?. Centrifuge isolated TEFF and TREG at
Centrifuge: 1200 34, 5 min
?. Resuspend TEFF cells in  of SOLUTION- 4 and TREG cells in  of SOLUTION- 4.Count them under microscope or Cellometer machine, according to the appropriate procedure (see step 2 of this protocol).
1 mL
0.2 mL
?. OPTIONAL STEPSIf required, it is possible to check the purity of isolated TEFF and TREG. Proceed as follows:Centrifuge at Remove the supernatant and resuspend the pellet in PBS 1X ;Add the adequate antibodies such as: CD4 APC-Cy7 (, BD cat. n. 557871), CD25 PE (, Miltenyi cat. n. 120-001-311) and CD127 AF647 (, BD cat. n. 558598) or conjugated to other fluorochromes;Incubate for , in the dark at RT; Wash with  of PBS 1X and centrifuge ;Resuspend the pellet in  PBS 1X and leave on ice until FACS acquisition with an appropriate protocol.
Centrifuge: 1200 34, 5 min
50 µl
2.5 µl
10 µl
10 µl
1 mL
Centrifuge: 1200 34, 5 min
350 µl
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