Dear Anna!

Please you look at the protocol:

Article Interactions of allogeneic human mononuclear cells in the tw...

Two-way MLC

Two-way MLC were set up using various types of cells, i.e. whole blood mononuclear cells, CD2+ cells, CD2+CD16− cells, CD2+ CD4− cells, CD2+ CD8− cells, or cells preactivated by OKT3 stimulation. A total number of 2 × 107 cells was cultivated in cell culture flasks in 20 ml of culture medium with or without 20 U/ml IL-2. Medium was exchanged when its colour changed to yellow, usually once or twice per week. Initial cell ratios between the two populations of allogeneic cells were variable, as indicated in the respective experiments. Cell counting error in the 50:50 setup was < 3%. Cell cultures were analysed for composition of cell populations and subsets at various time points using 21 days as end point.

For proliferation assays and determination of cell numbers after different periods of culture, 2 × 105 cells/well were cultured in 200 μl culture medium in triplicates using 96-well U-shaped microtitre plates. In the one-way MLC, 105 cells, ‘responder’ cells, were mixed with 105 irradiated (30 Gy) allogeneic ‘stimulator’ cells; in the two-way MLC, 105 cells of each of the two allogeneic populations were mixed. Following 2, 4, 6, 8, 10 and 14 days of culture 3H-thymidine (0.5 μCi/well, 5 Ci/mmol; NEN, Dreieich, Germany) was added and plates were incubated for 8 h. Cells were then harvested with an automatic cell harvester (Pharmacia LKB, Uppsala, Sweden). Activity was measured in a liquid scintillation counter (LKB Wallac, Turku, Finland). The data are given as mean ct/min of triplicates ± s.d.

Article Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction

Mixed lymphocyte reaction Patient and healthy control peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). Responder and stimulator cells were labeled with proliferation dyes (CellTrace CFSE and CellTrace FarRed, respectively) according to manufacturer protocol (Thermo Fisher Scientific, Waltham, MA, USA). Isolated PBMCs were stained with their corresponding proliferation dye at a 1:1000 dilution and incubated in the dark at 37˚C for 20 min. After incubation, PBMCs were washed in culture media (Roswell Park Memorial Institute [RPMI] 1640 medium; Mediatech Inc., Manassas, VA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at five-fold volume of the volume used for staining and incubated in the dark at 37˚C for another 5 min. Stained PBMCs were centrifuged and resuspended in culture media (RPMI containing 10% FBS) at a volume resulting in a final concentration of 1 £ 106 per mL. In the one-way MLR, stimulator cells were irradiated at 25 Gy. Responder and stimulator cells were co-cultured in 96-well plates at a concentration of 1 £ 105 cells per 100 mL each in RPMI media containing 10% FBS for 6
12 days. On days 6 and 12, 100 mL of the responder/stimulator mixture was removed for analysis using flow cytometry. In the two-way MLR, neither cell population was irradiated [15]. In two-way MLRs using normal healthy controls, both cell populations were plated at a concentration of 1 £ 106 per mL for a total of 2 £ 105 cells per well in 96-well plates unless otherwise noted. In two-way MLRs using patient samples, patient PBMCs were labeled with a single proliferation dye (CellTrace CFSE) and plated at 3 £ 105 cells per well (no addition of donor and recipient cells). The patient PBMC amounts per well were not adjusted by donor chimerism in each patient. In parallel, PBMCs from a single normal healthy control (without mixture of another control cells) were also cultured to evaluate baseline lymphocyte proliferation in the same condition. A unique healthy control without cryopreservation was used in two-way MLR for patient samples, who was not the donor used in the original transplant. Flow cytometry For surface marker analysis, isolated PBMCs were labeled with antibodies against CD33 (clone AC104.3E3, Miltenyi Biotec, Bergisch Gladback, Germany), CD20 (clone L27, Becton Dickson, East Rutherford, NJ, USA), CD14 (clone TUK4, Miltenyi Biotec), CD8 (clone BW135/80, Miltenyi Biotec) or CD4 (clone M-T466, Miltenyi Biotec) for 30 min in the dark at 4˚C. A non-stained control was used to differentiate between positively labeled cells. Analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson). For analysis of lymphocyte proliferation, a forward-scatter/side-scatter plot was used to gate the lymphocyte population. Non-proliferating and proliferating cells were captured in separate gates, and the percentages within each region was estimated (supplementary Figure 1). The proportion of living and dying cells was determined using the percentage of each cell population captured in either the combination of gates 1 and 2 or gate 3 (supplementary Figure 1) after 12 days in MLR culture. (attached file)