Lsrii sorp flow cytometer

The killing activity of HA_533-541-specific CD8+ T cells induced by the different vaccines was determined in vivo. Splenocytes from naïve BALB/c mice were loaded with cognate 5 μM HA_533-541 peptide (IYSTVASSL, JPT) or 10 μM HIV gag_199-207 control peptide (AMQMLKETI, JPT) and after 1 h the cells were loaded with CFSE (LifeTechnology) or CMTMR (LifeTechnology), respectively. Splenocytes (10⁶) were adoptively transferred into the tail vein of recipient mice 9 days or 5 - 6 weeks after the first or second immunization with SAM-H1/CNE, aMIV or sterile PBS. Mice were sacrificed 20 h after the adoptive transfer, and splenocytes were stained with live/dead yellow (LifeTechnologies) and frequencies of CFSE+ or CMTMR+ cells were measured by flow cytometry on a LSRII SORP flow cytometer (Becton Dickinson). The target cell specific lysis was calculated as % specific lysis = [1-(naïve mice/vaccinated mice)]x100 (39 (link)).

eGFP⁺ cell lines were cultured in serum + 2i medium for 2 weeks before flow cytometry measurements. eGFP levels of individual cell lines were measured on the BD LSRII SORP flow cytometer using BD High Throughput Sampler (HTS), which enabled sample acquisition in 96-well plate format. Measurements were repeated three times for each clone. Mean eGFP fluorescence intensities were calculated for each clone using FlowJo and all three replicates were averaged.

0.15–0.4 × 10⁶ cells were seeded in 96-well plates (Sarstedt). Samples were incubated for 5–10 min with 5 pg/mL propidium iodide (PI, Sigma Aldrich) before detecting dead cells (PI⁺). Proliferation of viable T-cells was determined by the reduction of CFSE (CFSE^low) and Lm infection rates in viable hMDMs were assessed by DsRed- or EGFP-expressing Lm as previously described (30 (link)). For surface expression analysis, cells were stained with fluorescent-labeled Abs (Table S1 in Supplementary Material) or corresponding isotype controls as defined by the manufacturer. Intracellular analysis required fixation with 4% paraformaldehyde (Sigma Aldrich) for 10 min on ice and permeabilization with 0.5% saponin (Sigma Aldrich) prior to staining. Results were recorded using a BD LSR II SORP flow cytometer (BD Bioscience) and analyzed by FlowJo software (Tree Star). Gating strategies are depicted in Figure S2 in Supplementary Material. The mean fluorescence intensity (MFI) of specific markers was normalized to the respective isotype control by division and is presented as relative fluorescence intensity (RFI). For T-cell proliferation and Lm infection rates, values of the respective controls were subtracted for each donor and condition, respectively, which allowed donor-specific evaluation of different treatments.

Cells from BM, spleen, blood, and peritoneal cavity were collected as described above and cell viability and total cell numbers were assessed by trypan blue staining using a Corning CytoSMART cell counter. Cells were preincubated with mouse-Fc block and viability dye (Zombie Aqua, BioLegend) in Mg²⁺- and Ca²⁺-free PBS for 15 minutes, before staining with fluorescently conjugated antibodies (Supplemental Table 1) prepared in washing buffer (WB: 2 mM EDTA, 0.02% sodium azide; 0.5% FCS, calcium/magnesium-free PBS) for 45 minutes. Samples were rinsed 3 times in WB and fixed with 10% formalin neutral buffered solution (Sigma-Aldrich), washed, and resuspended in WB for processing with a BD LSRII SORP flow cytometer/DIVA software. Postacquisition analysis was performed using FlowJo 10.6.2 (BD).