Cytoflex instrument

The establishment of long-lasting immunity and the subsequent protection against Leishmania parasites is dependent on the production of specific memory and effector T cells. To analyze the lymphocyte population, spleens from vaccinated and infected animals were obtained. Briefly, splenocytes were processed as described below to obtain a single-cell suspension. Subsequently, cells were counted, and 10⁶ cells/mL were incubated in the presence of 25 μg/mL of specific L. infantum or L. major SLAs at 37 °C, 5% CO₂ for 24 h. Cells were then triple-stained with anti-CD4 FITC (Clone GK1.5, Biolegend) or anti-CD8 FITC (Clone 53-6.7, Biolegend) and anti-CD44 (Clone IM7, Biolegend) and anti-CD62L (Clone MEL-14, Biolegend) for 30 min extracellular staining at 4 °C in the dark. Cells were then fixed with paraformaldehyde 4% w/w. Dead cells were excluded using a LIVE/ DEAD Zombie NIR Fixable Viability Kit (Biolegend). Cells were analyzed on a CytoFLEX^® instrument (Beckman Coulter, Life Science, Indianapolis, IN, USA), and the CytExpert™ software package version 2.5 (Beckman Coulter, Life Science, Indianapolis, IN, USA) was used for analysis based on at least 100,000 events per sample.

Cells were plated in 6-well plate with 1*10⁵ cells/well. After 12 hours, cells were infected with lentivirus vectors. After 48 hours incubation, cells were harvested, washed with PBS and incubated with Annexin V and PI, using the Annexin V-APC apoptosis detection kit (KGA1022, KeyGen, China). The flow cytometry analyses were performed with CytoFLEX instrument (Beckman Coulter, USA).

In this study, flow cytometry was employed to analyze various cell lines, including NCI-H23, JIMT-1, Calu-6, NCI-H358, and Jurkat. Adherent cells were washed twice with PBS and subsequently detached using Accutase (Sigma) at 37 °C. The cell suspension was transferred to a 50 mL conical tube, and FACS buffer (PBS containing 2% FBS) was added in a 2:1 v/v ratio (FACS buffer:cells). Cells were pelleted by centrifugation at 250×g and resuspended in FACS buffer at 4 °C. A total of 5.0 × 10⁵ cells/well were dispensed into 96-well u-bottom plates (Costar, Cole-Parmer Canada Company, Montreal, CA). Pre-diluted antibody samples were added to the wells to achieve a final volume of 100 µl/well and concentrations ranging from 0 to 14 nM (5-point dilution series), followed by a 30-minute incubation at 4 °C. Cells were subsequently washed twice by centrifugation at 250×g, with supernatant removal by aspiration, and resuspension in 200 µl FACS buffer at 4 °C. Detection reagent, anti-human Fc AlexaFluor488, was added, and samples were incubated at 4 °C for 20 min. Cells were washed twice in 200 µl FACS buffer, and flow cytometric analysis was performed on a CytoFLEX instrument (Beckman Coulter, Brea, CA). Median fluorescence intensity was determined for 20,000 live cells per sample.

Exosomal release was assessed by detecting exosomal markers such as CD81 and HSP70 using Western blot analysis and flow cytometry. Cell culture media were used to obtain exosomes. Exosomes were prepared using standard differential centrifugation in the following manner: centrifugation at 3000 × g for 20 min at 4℃ to obtain plasma, then 10,000 × g for 20 min at 4℃ to remove cells and platelets, and then twice at 100,000 × g for 70 min at 4℃ with a SW‐41 rotor, followed by washes with phosphate‐buffered saline (PBS). PBS was used solely for vehicle control. A total of 5 µg of exosomes was incubated for 15 mins with 1.25 µl aldehyde/sulphate latex beads, 4% w/v (4 µm, A37304; Invitrogen), and then incubated with anti‐CD81 (ab219209; Abcam) and anti‐Hsp70 (ab183435; Abcam). After fixing with 1% PFA, flow cytometry was performed with a FACSCalibur flow cytometer (BD Biosciences). The results were analysed by a CytoFLEX instrument (Beckman Coulter).