Symphony a5

Cell migration was measured using a 24 well-format Transwell migration chamber (pore size, 5 µm, Corning, 3421). NK cells were isolated from splenocytes using Stem Cell Technologies NK isolation kit (19855) as described by manufacturer. First, NK cells were suspended in fresh medium (RPMI, 10% FBS, pen/strep) supplemented with 20 ng/mL IL-2 (PeproTech, 212-12) and seeded on 96-well plates (1 × 10⁶ cells/well). After 24 h, cells were washed with PBS and suspended in migration buffer (RPMI, 0.2% FBS) at 1 × 10⁶ cells/mL. For the migration to CCL5 chemokine, the migration buffer supplemented with 100 ng/mL CCL5 (PeproTech, 500-P118) was added in the lower compartment and 100 µl of cell suspension was added into each insert. After 4 h at 37 °C, the sample from lower compartment was collected and stained for NK1.1, NKp46 and Live/Dead marker (as described in Flow Cytometry) together with input sample. Samples were then suspended in equal volume and equal volume of sample was acquired using the BD High Throughput Sampler combined with BD Symphony A5 and the number of alive NKp46+, NK1.1+ positive cells per sample was analyzed. Migration rate was determined as a (nb of cells from sample / nb of cells from input sample) x 100%.

Cell phenotyping was performed by flow cytometry on 23 fresh PBMC samples from young donors and 78 fresh PBMC samples from older donors. For each staining, 1 × 10⁶ PBMCs were used. Lymphocytes were gated based on FSC/SSC profile and doublets/dead cell exclusion.
Absolute cell count was performed by flow cytometry on freshly collected blood of ten healthy donors and six HAART HIV-infected patients. For each staining, 100 μL of blood was used in Trucount tubes. After doublets/dead cell exclusion, lymphocytes were gated based on FSC/SSC profile and CD45 expression.
The antibodies are listed in Supplementary Table 3. Flow cytometry was performed on an LSR Fortessa Cell Analyzer (BD Biosciences), and automatic compensation was applied.
We used BDSymphony A5 (BD Biosciences) to perform high-dimensional single cells immunophenotyping and characterize the heterogeneity of naive and T_SCM CD4 cells from 2 million frozen PBMCs.
Flow cytometry of HIV participants was performed on a BD FACS Celesta (BD Biosciences) at University of Malaya and automatic compensation was applied.

Samples were acquired on a BD Symphony A5 instrument. Standardized SPHERO rainbow beads (Spherotech) were used to track and adjust photomultiplier tubes over time. UltraComp eBeads (Thermo Fisher) were used for compensation. Up to 5 × 10⁶ cells were acquired per sample. Data were analyzed using FlowJo v10 (BD Bioscience). For Boolean analysis of variant cross-binding, data were imported into SPICE 6 [NIH Vaccine Research Center (55 (link))]. Cell sorting was performed on a BD FACS Aria II instrument in low pressure mode using a 70 μm nozzle. Cells were sorted into DNA LoBind Eppendorf tubes containing cell lysis buffer (Qiagen).

Staphylococcus aureus (Wood strain without protein A) BioParticles conjugated with Alexa Fluor 488 (Thermo Fisher, S23371) were reconstituted in tissue culture-grade PBS at 20 mg/mL. Prior experiment, the 50-fold dilution was prepared with the tissue culture-grade PBS. BioParticles in volume of 25 µL were injected into wound margins of WT and HIF-1α KO mice at day 4 post-wounding. Mice were sacrificed 45 min post injection and single-cell suspension from spleen was analyzed by flow cytometry. Each splenic single-cell suspension was prepared in equal volume and equal volume of sample was acquired using the BD High Throughput Sampler combined with BD Symphony A5, followed by the analysis of circulating particles per spleen.