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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Magnetic bead-based TREG-TEFF cell isolation from PBMC with Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit, list of published work using this procedure:</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. Isolate PBMCs according to the standard protocol "Separation and purification of human PBMC from BUFFY COAT" or "Separation and purification of human PBMC from FRESH BLOOD"
?. 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 complete medium 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 Miltenyi Buffer (for 10x106 cells).
100 µl
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?. Add  of CD4+T Cell Biotin-Antibody Cocktail (for 10x106 cells).
10 µl
?. Mix well and incubate for  at
4 °C
?. 8.     Add  of Anti-Biotin MicroBeads (for 10x106 cells), mix well and incubate  at
20 µl
4 °C
?. Add  of cold Miltenyi Buffer and centrifuge at
5 mL
Centrifuge: 1200 34, 5 min
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?. Discard the supernatant and resuspend the pellet in  of cold Miltenyi Buffer.
500 µl
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?. Place LD column in the magnetic field of suitable MACS Separator (violet).
?. Prepare column by rinsing it with  of Miltenyi Buffer (trash the effluent).
3 mL
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?. 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 Miltenyi Buffer and 1 last time with  of Buffer. Collect total effluent that is consisting of unlabeled pre-enriched CD4+ cell fraction.
3 mL
2 mL
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?. Centrifuge the obtained effluent at
Centrifuge: 1200 34, 5 min
?. Remove supernatant and resuspend cell pellet in of cold Miltenyi Buffer (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.
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?. Add of CD25 MicroBeads (for 10x106 cells), mix well and incubate , at , in the dark.
10 µl
4 °C
?. Add of cold Miltenyi Buffer and centrifuge at
5 mL
Centrifuge: 1200 34, 5 min
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?. Remove the supernatant and resuspend the cell pellet in of cold Miltenyi Buffer.
500 µl
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?. Place the MS column in the magnetic field of a suitable MACS Separator (green).
Magnetic separation: Positive selection of CD4+CD25+T regulatory cells.
?. Prepare the column by rinsing with of cold Miltenyi Bufferand trash the effluent.
500 µl
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?. 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 Miltenyi Buffer.
2 mL
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 Buffer 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
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?. 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 RPMI/10%FBS and TREG cells in  of RPMI/10%FBS.Count them under microscope or Cellometer machine, according to the appropriate procedure (see step 2 of this protocol).(See this link for RPMI-FBS 10% recipe)
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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?. 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.Mix 10 µl  of cell suspention with an equal amount of Türk solution (dilution factor = 2), allow mixture 3 min at room temperature.Take 10 µl of the mixture and place it inside a Bürker chamber and view under an optical microscope using 40X magnification.Count the cells in each square found in the four corners and in the central square (see figure 1 below), including thosethat lie on the bottom and left-hand perimeters, but not those that lie on the top and right hand perimeters (see figure 2 below).Total number of cells per ml = mean number of cells x dilution factor x 104 (hemacytometer volume).Figure 1The gridded area of the chamber consists of nine 1 mmq squares. These squares are subdivided in three directions; 0.0625 mmq, 0.05 mmq and 0.04 mmq. The central square here in Figure 1 is further subdivided into 0.0025 mmq = 1/25 mmq squares. Count cells in 5 squares as shown.Figure 2Concerning those cells that lay on the perimeter of the square, count following this scheme.For automatic cell count with Cellometer machine use Trypan Blue. The machine will calculate the n°of cells/ml and the % of viability.Take 10 µl of cell suspention and add an equal amount of Trypan Blue. Use all the volume to place it in a counting chamber. Place the chamber inside Cellometer and count.
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