Fluorochrome conjugated antibodies

Lymphocytes in whole blood were surface stained with fluorochrome-conjugated antibodies according to the manufacturer’s instructions (BioLegends, San Diego, CA, USA). Mice whole blood (100.0 μL) was pre-treated with 1.0 μg of purified anti-mouse CD16/32 (BioLegend, San Diego, CA, USA) to block Fc-receptors, followed by cell surface staining with 0.125 μg of Brilliant Violet 421-conjugated anti-mouse CD3, 0.125 μg of APC/Fire 750-conjugated anti-mouse CD4, 0.25 μg of Brilliant Violet 510-conjugated anti-mouse CD8a, 0.0625 μg of APC-conjugated anti-mouse CD19, 0.15 μg of Brilliant Violet 605-conjugated anti-mouse CD25, 0.125 μg of PerCP/Cy5.5-conjugated anti-mouse CD335 and 0.5 μg of PE-conjugated anti-mouse FOXP3 antibodies (BioLegend, San Diego, CA, USA). After cell staining, red blood cells were lysed with 2.0 mL of RBC lysis buffer (BioLegend, San Diego, CA, USA), and stained cells were acquired using Novocyte^TM flow cytometer (ACEA Biosciences, San Diego, CA, USA). The data were analyzed using NovoExpress^® software version 1.3.0 (ACEA Biosciences, San Diego, CA, USA). Data were expressed as mean percentage ± standard deviation of three biological replicates, and Student’s t-tests were performed with a p-value ≤0.05 (*) representing statistically significant differences.

After 48 h of treatment, the cell co-cultures were incubated in Accutase (at 37 °C for 15 min) to dissociate huTGOs into single cells as previously published [30 (link)]. All cells were collected by centrifuging at 300× g for 5 min, then resuspended in 100 µL of diluted Zombie UV dye (1:100 in PBS) and stained for 15 min at RT. The cells were incubated at 4 °C for 30 min with fluorochrome-conjugated antibodies specific for CD8a, CD14, CD15, CD11b, CD33, CD137, EpCAM, granzyme B, Perforin, HLA-DR, and PD-L1 (1:100 dilution, all from BioLegend), diluted in 100 μL cell staining buffer. Cells were washed with cell staining buffer (BioLegend) and incubated with the Cytofix/Cytoperm Fixation/Permeabilization Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 20 min at 4 °C. Cells were then washed and resuspended in 100 µL of cell staining buffer and stained at 4 °C for 30 min with fluorochrome-conjugated intracellular antibodies specific for perforin, IL-2 and interferon-gamma (IFN-g) (both from BioLegend) diluted in 100 µL cell staining buffer. Cells were washed, resuspended in 300 µL of cell staining buffer, filtered, and then analyzed on an LSRII system (BD Biosciences). An unstained cell sample and single stained beads for each antibody were used as gating controls. Data were analyzed using FlowJo software (BD Biosciences).

BALF cells, mLN, or lung single cell suspensions were resuspended in cell staining buffer (no. 420201, BioLegend, San Diego, CA, USA) and blocked with TruStain FcX (anti-mouse CD16/32) antibody (no. 101320, BioLegend, San Diego, CA, USA) for 10–15 min on ice. The cells were then incubated 30 min with the following specific fluorochrome-conjugated antibodies or isotype controls (BioLegend, San Diego, CA, USA ) in the dark at 4 °C: Alexa Fluor-488 anti-CD45, PE anti-CD3, APC anti-CD4, FITC anti-CD4, PE-Cy7 anti-CD8α, APC anti-glycosylated CD43 (1B11), FITC anti-CD43, FITC anti-CD44, APC anti-CD11a, PE/Cy5 anti-CD69, Pacific Blue anti-CD54, APC anti-CD11b, PE anti-Ly6G, or FITC isotype control antibodies. PE-conjugated tetramer specific for H-2Db IAV NP_366–374 (ASNENMETM) was from MBL International Corporation (Woburn, MA, USA). After washing, stained cells were acquired in an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA USA). Cell death was determined by flow cytometry using APC annexin V apoptosis detection kit with propidium iodide (no. 640932, BioLegend, San Diego, CA, USA). Flow cytometry data were analyzed by using FlowJo software v10.6.2.

Spleen cells were obtained from non-immunized mice, self-cured mice 25 days post P. yoelii 17X infection and immunized mice with rexPy or rexC on day 20 after the second immunization, as described above. Splenocytes were analyzed for the expression of different markers with an LSRFortessa flow cytometer and data were analyzed with FlowJo software. T cells subsets were identified according to lymphocytes' SSC-A/FSC-A profile and labeling with CD4-PerCp or CD8-Alexa Fluor-700 antibodies (Biolegend). The phenotype of CD4⁺ and CD8⁺T cells in spleen was analyzed with a panel of fluorochrome-conjugated antibodies obtained from Biolegend that included: CD62L-Pacific Blue, PD1-PE/Cy7, CD127-PE and CD44-FITC.