Lsr flow cytometer

Cell-binding assays to calculate monomeric affinity were modified versions of “Cytometry Cell Binding” protocol above. Recombinant SARS-CoV-2 spike protein S1 C-terminal domain was dialyzed into PBS (Millipore #71505) and then covalently conjugated to R-phycoerythrin using a commercial amine coupling kit (Abcam #ab102918). Transfected HEK293 cells were transferred to v-bottom 96-well plates (Greiner #651261). Comparatively small quantities of cells were used (10 μL, or about a few thousand cells per well) in order to ensure that the assumptions around free ligand concentration for the 1:1 binding model equation were not violated [81 (link)]. After the initial wash and DAPI stain, cells were resuspended in a 3× dilution series of spike protein going across the wells of the plate, starting at 16.7 μM diluted in 1% BSA in PBS with calcium and magnesium ions (Gibco #14040133). Binding was allowed to reach equilibrium over an hour at 4°C before two washes in PBS. For washes, cells were centrifuged at 300 × g for 7 minutes at 4°C, then supernatant was carefully aspirated by pipette. Cells were finally resuspended in 40 μL 1% BSA PBS and measured with a BD LSR flow cytometer.

Gating schemes are provided in the Supplementary Information. Unless otherwise indicated, cytometry was performed using an LSR Flow Cytometer (BD Biosciences), and analysis was performed using FACSDiva version 9, CellQuest version 6 and FlowJo version 9 software (BD Biosciences).

CH12 cells were cultured for 72 h in the presence of TGF-β (1 ng/ml; R&D Systems Europe), IL-4 (5 ng/ml; Peprotech), and an anti-CD40 antibody (200 ng/ml; eBioscience). Cells were then stained with an anti-IgA-PE antibody (Southern Biotech) to assess CSR efficiency by flow cytometry. Before analysis, DAPI was added to discriminate dead cells. Samples were analyzed using an LSR flow cytometer (BD Biosciences) and FlowJo software.

We used thymidine double blocks to synchronize HPV16 positive cervical carcinoma cell line (SiHa): after a first 24 hours thymidine (2.5 mM) block, the cells were released for 2 hours and infected with the recombinant adenoviruses expressing GFP-16E2, GFP-TAD GFP-DBD and GFP as control, followed by a second thymidine block. The cells were released 22 h later for cell cycle analyses.
A549 cells were treated with 2.5 mM thymidine for 24 h and infected with recombinant adenoviruses expressing GFP or the GFP-16E2 fusion protein half way through the thymidine treatment. After which the cells were released from thymidine block and harvested for analysis at the indicated time points (from T0 to T16). For synchronization in metaphase, cells were treated with thymidine for 24 h and then released for 16 h in the presence of 100 ng/ml nocodazole (NR0), nocodazole was then removed and cells were harvested 5 hours later (NR5 corresponding to 5 h post nocodazole release) for cell cycle analysis. The cells were fixed in 70% ethanol before flow cytometry to detect DNA content using 0.5 μg/ml DAPI and a 1:500 dilution of an anti-H3p antibody (Ab7031, Abcam). Cell cycle analyses were carried out using an LSR flow cytometer (BD Biosciences, USA).

Adipocytes and adipose tissue macrophages (ATM) were isolated from lipopolysaccharide (LPS) or PBS‐injected mice for flow cytometry by collagenase digestion as previously described 18, 19. Used antibodies are found in Table S2. ATMs were stained for CD45, CD11b and F4/80. Adipocytes were stained for CR4. Fluorescence minus one controls were included for both cell types. Samples were measured on a LSR flow cytometer (BD Bioscience, San Jose, CA, USA) and the data were analysed using FlowJo (FlowJo LLC, Ashland, OR, USA).

Cytosolic Ca²⁺ concentrations were measured as described previously (Baba et al., 2006 (link)). In brief, splenocytes were loaded with indo-1 acetoxymethylester (Indo-1 AM) and Pluronic F-127 (Invitrogen) and stained with antibodies to B220 and Igκ. Cells were stimulated with 10 μg/ml NP-Ficoll (Biosearch Technologies). Changes in fluorescence intensity were monitored on an LSR flow cytometer (BD Biosciences).