Round bottomed 96 well plate

Spleens were harvested from each mouse on the indicated day post-sensitization. Mononuclear cells were isolated with Histopaque-1083 (Sigma). Cells were resuspended at 1 × 10⁶ cells/ml in complete media [RPMI-1640 media (Mediatech, Manassas, VA, USA) supplemented with 1% L-glutamine (Mediatech), 1% antibiotics (Mediatech), 50 μM 2-mercaptoethanol (Sigma) and 10% Cosmic calf serum (Hyclone, Logan, UT, USA)]. Next, 100 μl of cells were added to each well of a 96-well round-bottomed plate (Corning, Corning, NY, USA). PLP_139–151 in 100 μl of complete media was added into culture in a dose dependent manner. Cells were incubated at 37°C, 5% CO₂ for the indicated times in the presence of the indicated peptide doses. Anti-IGF-1R antibody (αIR3) (Millipore, Marlborough, MA, USA) was added into the PLP_139–151-stimulated spleen cell cultures at a final concentration of 1 μg/ml. Eighteen hrs prior to harvesting cultures, the cells were pulsed with 1 μCi/well of tritiated thymidine (³H-TdR) (PerkinElmer, Boston, MA, USA). The cells were harvested onto glass fiber filters (PerkinElmer) for measurement of radiolabel incorporation using a liquid scintillation counter (PerkinElmer).

The biofilm mass of sixteen MAB gene knockout clones and reference MAB 19977 was measured with the crystal violet staining method. Bacteria grown in the mid-log phase were resuspended at a concentration of 1 × 10⁸ CFU/mL in the Synthetic Cystic Fibrosis Sputum Medium (SCFM) established and prepared as previously described [28 (link)]. The uniformity for all the tested groups was confirmed with OD readings at OD₆₀₀. A volume of 100 μL was placed in columns of a 96-well round-bottomed plate (Corning, Corning, NY, USA) for both the experimental and control groups (eight replicates) and incubated for one week at 37 °C (no agitation). The biofilm formation was quantified at 7 days by removing the supernatant from all wells and adding 125 μL of 0.1% crystal violet (CV) solution for 10 min at room temperature. After, the plates were rinsed three times with the distilled water and left to dry for 10 min. CV-stained biofilms were then solubilized with 125 μL of 30% acetic acid for 10 min. The samples were moved to new clear 96-well flat-bottomed plates, and the optical density was measured at OD₅₇₀ in a plate reader (Epoch Microplate Spectrophotometer, BioTek, Winooski, VT, USA). The experiment was performed in three biological replicates.

Purified CD4⁺CD25^– cells (responder cells) were either directly plated or preloaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then seeded into 96-well round-bottomed plates (Corning Costar Corp, NY) in triplicate at a density of 5 × 10⁴ cells/well. Cell proliferation was induced by stimulation with anti-CD3 (1 μg/ml) in the presence of irradiated allogeneic PBMCs as APC (2×10⁵/well, irradiated at 35 Gy) for 4 days. Proliferation was measured either by monitoring CFSE dilution or by thymidine incorporation, in which ³H thymidine (0.5 μCi/well, Amersham, Freiburg, Germany) was added to the culture 10 hours prior to cell harvesting.
To observe the inhibitory effect on cell proliferation, CD25⁺CD3⁺CD56⁺ cells directly purified from TILs, FOXP3-transduced CD3⁺CD56⁺ cells, TGF-β1-treated CD3⁺CD56⁺ cells, or CD4⁺CD25^high conventional Treg cells purified from PBMC were added to the assays at different ratios to responder cells from the same donor. Relative suppression was calculated as: % suppression = [1 – % of proliferation in coculture/% of proliferation in responder cells culture alone] × 100%. In some settings, the cells to be tested were separated from responder cells using transwell (0.4 μm, Corning Costar Corp). In other settings, exogenous rhIL-2 was added to the culture at a concentration of 10 ng/ml.

CD11c^hiMHCII^intCD11b^hi sorted by using a cell sorter FACS Aria I (Becton Dickinson) from DLNs and tumours from untreated and treated mice. DCs (3x10³) and CellTrace violet (CTV) labeled CD8 T cells (3x10⁴) sorted from normal mice LNs were cultured for 3 days in 96 well round-bottomed plates (Costar) in RPMI 1640 medium supplemented with 5% FCS, penicillin (100U/ml), streptomycin (100μg/ml) and 50 μM 2-ME. Sorted CD4⁺CD25^- T cells were added to the culture separately. Anti-CD3 mAb (145-2C11) at a final concentration of 0.1 μg/ml was added to the cultures for stimulation. CTV dilutions were used to determine the degree of CD8 T cell proliferation. After 3 days, cells were collected, treated with 5 mM EDTA and stained with anti-PD-1 mAb.

In order to determine the immunosuppressive function of MDSCs, the sorted MDSCs were co-cultured with CFSE-labeled splenic CD4⁺ T cells in the study. Splenic CD4⁺ T cells were stained with fluorescent dye CFSE (5 μM, Invitrogen) for 10 min at 37°C protected from light. RPMI 1640 medium (Gibco, Carlsbad, CA) containing 10% fetal calf serum (Gibco, Carlsbad, CA) was added to wash the cell pellets for 3 times. Sorted MDSCs were co-cultured with CFSE-stained CD4⁺ T cells at a ratio of 1:1 in 96-well round-bottomed plates (Costar, Corning, NY) in the presence of anti-CD3 mAbs and anti-CD28 mAbs (Biolegend, San Diego, CA). The cells were incubated in RPMI 1640 medium supplemented with 10% fetal calf serum at 37°C in a humidified 5% CO2 atmosphere for 72 h protected from light. The proliferation of CD4⁺ T cells was detected by flow cytometry at 488 nm excitation light to determinate the suppressive activity of MDSCs.