TITLE: Magnetic bead-based CD4+ T cell isolation from PBMCs with Dynabeads: CD4 Positive 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 works using this protocol:</div><div class = "text-block"><span>- 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. </span><a href="https://doi.org/10.1186/s12974-018-1248-8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://doi.org/10.1186/s12974-018-1248-8</span></a><span></span></div><div class = "text-block"><span>- 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. </span><a href="https://doi.org/10.1038/srep33738" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/srep33738</span></a></div><div class = "text-block"><span>- 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. </span><a href="https://doi.org/10.1016/j.humimm.2015.09.032" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1016/j.humimm.2015.09.032</span></a></div></div>

[STEPS]
?. Isolate PBMCs according either to the standard protocol from fresh blood or from buffy coat.
?. 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. The machine will calculate the n° of cells/ml and the % of viability.- Take 10 µl of cell suspension 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.For manual cell count use Türk solution for checking purity.- Mix 10 µl  of cell suspension with an equal amount of Türk solution (dilution factor = 2), allow mixture 3 min at room temperature.- Take 10 µlof 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).
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?. 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 of Buffer 1 (found in the kit materials), 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.
2 ml
?. Repeat the washing step 2 or 3 times to make sure that DMSO is all washed up.
?. After counting, centrifuge PBMCs sample at .
Centrifuge: 1200 34, 5 min
?. Discard supernatant and resuspend pellet of 10x106cells in  of Buffer 1.
1 ml
?. Transfer cell suspension into the tube with beads, and resuspend vigorously.
?. Incubate the beads with cells for  at  with gentle rotation by putting the Sample Mixer in the fridge.
4 °C
?. 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  of Buffer 1 and resuspend the cells very vigorously because of aggregates.
2 ml
?. 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  Buffer 2. [The volume is calibrated for 10x106cells, for lower or higher number of cell resize the volume accordingly].
100 µl
?. Add  of DETACHaBEAD® CD4 for each 10x106 PBMCs.(Resize this volume if the number of starting cell is different)
10 µl
?. Add another  of Buffer 2 to increase the volume and transfer everything in a 1.5 mL eppendorf.
500 µl
?. Incubate  at (RT) with gentle rotation by using a Sample Mixer.
4 Room temperature
?. 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  Buffer 2 and collect the supernatant each time.
500 µl
?. Add to detached cell suspension Buffer 2 to a final volume of 10 mL and centrifuge at .
Centrifuge: 1200 34, 5 min
?. Resuspend the cells for cell counting in : follow the appropriate protocol (see step 2 of this protocol). Check the viability with Trypan blue.
1 ml
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?. OPTIONAL STEPCheck 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.
?. EXPECTED RESULTS
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 CoatIf checked, purity of the isolated CD4+ cells must be ≥95 %