Streptavidin phycoerythrin pe

Expression of the various CD19-CARs on T cells was detected using either biotinylated anti-mouse FMC63 scFv monoclonal antibody (Biowan) followed by staining with streptavidin-phycoerythrin (PE) (BD Biosciences), or with an allophycocyanin (APC)-labeled anti-mouse FMC63 scFv monoclonal antibody (Biowan). 1 × 10⁶ cells were stained for cell surface markers to analyze T cell differentiation and exhaust status. The following antibodies were used: anti-CD3-FITC (BD Biosciences), anti-human CD3-APC (BD Biosciences), anti-CCR7-BV421 (BD Biosciences), anti-CD 45RO-APC (BD Biosciences), anti-CD4-BB515, anti-CD8-BV510 (BD Biosciences), anti-CD45RA-PE- Cy7 (BD Biosciences), anti-CD62L-PE (BD Biosciences), anti-PD-1-APC (BD Biosciences), anti-TIM-3APC (BD Biosciences), and anti-LAG-3-APC (BD Biosciences). Samples were analyzed on either FACSCanto II flow cytometers (BD Biosciences) or Novocyte3110 (AECE).

Topical 5% imiquimod cream (Aldara^™) was from 3M Health Care Limited (Loughborough, UK). TO-PRO-3 and RPMI-Glutamax, 5-(and-6)-carboxyfluorescein succinimidyl ester (CFSE) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Empty CyaA was produced in Escherichia coli as described previously [19 (link)]. CyaA was biotinylated with EZ-Link^® HPDP-Biotin (Thermo Fisher Scientific) and purified with a 7000-Da molecular weight cutoff Zeba^™ Spin desalting column (Thermo Fisher Scientific). Streptavidin-phycoerythrin (PE) was from BD Biosciences (Franklin Lakes, NJ, USA). Synthetic peptides HPV16 E7_49-57, HPV18 E7_AS43-49, and ovalbumin_257-264 were obtained from Millegene (Toulouse, France), 15-mer peptide banks (HPV16 E7 and HPV18 E7) overlapping by 11 amino acids were from Eurogentec (Seraing, Belgium). HPV16 E7_43-98 was synthesized by Genosphere (Paris, France).

PBLs were seeded into 24-well plates and incubated for 24 h with rIFNs or with media alone at 20°C. At this point, cells were collected and incubated for 1 h at 4°C with anti-IgM-FITC and anti-trout IgD-APC as described above. Following several washing steps, cells were resuspended in staining buffer and IgM⁺IgD⁺ B cells isolated by flow cytometry using a BD FACSAria III cell sorter (BD Biosciences) based on their FSC/SSC profile and then on the basis of the fluorescence emitted by the anti-IgM and anti-IgD antibodies. Approximately 70,000 IgM⁺IgD⁺ B cells were collected in PBS for subsequent RNA isolation.
To confirm a direct effect of rIFNs on IgM⁺IgD⁺ B cell survival, PBLs were incubated for 1 h at 4°C with a biotinyilated Fab fragment of anti-IgM 1.14 (to avoid cell activation) in staining buffer. Following two washing steps, Streptavidin-Phycoerythrin (PE) (BD Pharmingen) was added. After 20 min at 4°C, cells were resuspended in staining buffer and IgM⁺IgD⁺ B cells isolated as described above. Sorted IgM⁺IgD⁺ B cells were then incubated with rIFNs or media alone for 3 days at 20°C. After this time, cells were counterstained with 0.2 μg/ml DAPI, and analyzed on a FACS Celesta flow cytometer.

MAb specificity for HIV-1 proteins was evaluated on a panel of HIV-1 gp120/gp140 Env glycoproteins, the linear C1 region, and the third variable-loop (V3) peptide epitopes, and V1-V2 scaffolds were measured by a custom binding antibody multiplex assay, as previously described (6 (link), 60 (link), 61 (link)). Positive controls included HIVIG (provided by the NIH AIDS Reagent Program) and 7B2 IgA MAb titrations and single-point HIV-1 IgG MAb controls CH58_IgG1 (9 (link)), VRC01 IgG (62 (link)), CH106 IgG (63 (link)), 2G12 IgG (64 (link), 65 (link)), 17b IgG (66 (link)), and F105 IgG (67 (link)). Blank beads and HIV-1-negative sera were used as negative controls. HIV-1-specific antibody isotypes were detected with mouse anti-human IgA (BD Pharmingen) at 4 μg/ml, followed by washing and incubation with streptavidin-phycoerythrin (PE) (BD Pharmingen). Antibody measurements were acquired on a Bio-Plex instrument (Bio-Rad) with 21CFR Part 11 compliant software, and the readout was expressed as mean fluorescence intensity (MFI). Each test MAb was assayed at a starting concentration of 100 μg/ml and titrated by 2-fold dilutions. Results were reported as area under the curve (AUC) binding titers.

Cells were incubated with a biotin‐SP‐AffiniPure F(ab)’2 fragment‐specific goat anti‐mouse IgG antibody (Jackson ImmunoResearch) and then with streptavidin‐phycoerythrin (PE) (BD Biosciences). Then, the cells were incubated with anti‐PE microbeads at 4°C for 15 min, and magnetic separation was performed to collect CAR‐positive cells according to the manufacturer's protocol (Miltenyi Biotec).

CAR expression was assessed using biotinylated anti-Fc (Jackson ImmunoResearch, 1:100, RRID: AB_2337663) antibody followed by streptavidin-phycoerythrin (PE) (BD Biosciences, 1:20, RRID: AB_10053328) and by staining for the CD19t extracellular sequence with CD19-PE-Cy7 (BD Biosciences, clone SJ25C1, 1:100, RRID: AB_396893). Target lines were characterized by staining with IL13Rα2-PE (BioLegend, clone SHM38, 1:100, RRID: AB_11218806), IL13Rα1 (BioLegend, clone SS12B, 1:100, RRID: AB_2562552), and IL-4Rα-PE (BD Pharmingen, clone hIL4R-M57, 1:20, RRID: AB_394355). In other assays, additional antibodies were used as specified: CD107a-FITC (BD Biosciences, clone H4A3, 1:9, RRID: AB_396134), CD45 PerCP (BD Biosciences, clone 2D1, 1:20, RRID: AB_2868830), CD3-VioBlue (Miltenyi Biotec, 1:20, RRID: AB_2725961), CD8 APC-Cy7 (BD Biosciences, clone SK1, 1:50, RRID: AB_396892), and IFNγ-APC (BD Biosciences, clone B27, 1:100, RRID: AB_398580). For staining, cells were washed and resuspended in fluorescence-activated cell sorting (FACS) stain solution [Hank’s Balanced Salt Solution (HBSS), 20% (vol/vol) FBS, 0.1% (wt/vol) NaN₃ (CAS 26628-22-8)], incubated with antibodies for 30 min at 4 °C, followed by secondary stain if necessary, and then washed and run on the MACSQuant (Miltenyi Biotec, RRID: SCR_020268). Flow data were analyzed with FCS Express 4 (De Novo Software, RRID: SCR_016431).

HLA‐A*02:01‐GILGFVFTL (A2/M1₅₈) and HLA‐A*02:01/GLCTLVAML (A2/GLC) monomer (ImmunoID, University of Melbourne, Melbourne, VIC, Australia) was tetramerized with streptavidin‐phycoerythrin (PE) (BD Biosciences, San Jose, CA, USA). PBMCs from HLA‐A*02:01‐expressing donors were enriched for A2/M1₅₈‐ and A2/GLC‐specific CD8⁺ T cells using the A2/M1₅₈‐ and A2/GLC‐PE conjugated‐tetramers, respectively, followed by magnetic enrichment as previously described23, 24, 32 using anti‐PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched, unenriched and flow‐through samples were then stained for flow cytometry.