Probes for murine Ptpn6 (AF647 conjugate, Assay ID: VB1-3030134-PF) and murine beta-actin (AF750 conjugate, Assay ID: VB6- 12823-PF) were from ThermoFisher Scientific. Single cell suspensions of splenocytes were prepared and 2 × 106 cells were plated per well of a 96-well V-bottom plate. Cells were stained with LIVE/DEAD™ Fixable Aqua (ThermoFisher), blocked with anti-CD16/32 (BD Biosciences) and stained with the following antibodies to surface markers: Panel 1: NKp46 FITC, TCRβ PE-Cy7, CD25 PE, B220 BV421, CD8 BV650, CD4 BV711 or Panel 2: F4/80 FITC, Ly6g PE-Cy7, CD11c PE, CD11b e450, MHCII BV711 in Superbright staining buffer (ThermoFisher). Cells were fixed and permeabilized using PrimeFlow Assay Kit buffers following the manufacturer's instructions (ThermoFisher). Target probes were hybridized for 2 h at 40°C then cells were washed and stored overnight at 4°C in PrimeFlow RNA Wash Buffer with RNase Inhibitor 1. The following day, signal amplification steps were performed according to manufacturer's protocol and cells were analyzed by flow cytometry on a BD Fortessa flow cytometer. Data was analyzed using FlowJo (Treestar) and the mean fluorescence intensity (MFI) of the Ptpn6 mRNA probe was determined in the following cell types: PMNs (CD11b+ Ly6g+), Tregs (B220− TCRβ+ CD4+ CD25+ FoxP3+), CD4+ T cells (B220− TCRβ+ CD4+ CD8−), and CD8+ T cells (B220− TCRβ+ CD4− CD8+).
Super bright staining buffer
Manufactured by Thermo Fisher Scientific
Super Bright Staining Buffer is a laboratory reagent used to prepare samples for fluorescent staining and imaging. The buffer is designed to optimize the brightness and stability of fluorescent dyes, allowing for clear visualization of target analytes. It maintains the pH and ionic conditions necessary for effective staining without interfering with the fluorescent signal.
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Lab products found in correlation
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32, by BD (1 mentions)
Cd25 pe, by Thermo Fisher Scientific (1 mentions)
F4 80 fitc, by Thermo Fisher Scientific (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Fixable viability dye efluor 455uv, by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Facsdiva 8, by BD (1 mentions)
Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Human trustain fcx fc receptor blocking solution, by BioLegend (1 mentions)
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Ctl anti aggregate medium, by Cellular Technology (1 mentions)
Ctl test medium, by Cellular Technology (1 mentions)
Brilliant violet buffer, by BD (1 mentions)
Live dead fixable aqua dead cell dye, by Thermo Fisher Scientific (1 mentions)
Anti ly6g apc clone 1a8, by BioLegend (1 mentions)
Lsrii fortessa flow cytometer, by BD (1 mentions)
5 protocols using super bright staining buffer
1
Murine Ptpn6 and Beta-Actin Expression
2
Multiparametric Flow Cytometry of Immune Cells
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Single‐cell suspensions were stained with Fixable Viability Dye eFluor 455UV and anti‐CD16/CD32 (both from Thermo Fisher) in PBS prior to addition of the relevant fluorochrome‐conjugated antibodies in FACS buffer supplemented with Super Bright staining buffer (Thermo Fisher). The following antibodies were used: Life Technologies: CD11c‐SB436 (clone N418), F4/80‐eFluor 506 (clone AG BM8), Sca‐1‐SB600 (clone D7), CD11b‐SB780 (clone M1/70), CD86‐FITC (clone GL1), Ly6C‐PerCP‐Cy5.5 (clone HK1.4), CD68‐PE (clone FA‐11), MHC‐II‐PE‐eFluor 610 (clone IA/IE), CD206‐PE‐Cy7 (clone (MR6F3), CD64‐APC (clone AFS98), CD45‐Af700 (clone 104), CD3‐APC‐ef780 (clone 145‐2C1), NKp46‐APC‐ef780 (clone 29A1.4), CD19‐APC‐ef780 (clone HIB19; BD), Siglec‐F‐SB645 (clone E50‐2440) and Ly6G‐SB702 (clone 1A8). Samples were acquired on an LSRFortessa running FACSDiva 8 software (Becton Dickinson, Wokingham, UK). Data were analysed using FlowJo software (TreeStar; version 10.4.2) (Figure S4 ). For samples with fewer than 1,000 total eosinophils acquired, data were not used to compare Ly6G+ and Ly6G− subsets.
3
Multiparameter Flow Cytometry Immunophenotyping
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Cells were washed with PBS, resuspended in PBS at a concentration of 3.0×106/mL, and incubated with Human TruStain FcX Fc receptor blocking solution (BioLegend) and Live/DEAD Fixable Blue Dead Cell Stain (Invitrogen) according to the manufacturers’ specifications for 20 min at room temperature, protected from light. The cells were then washed once in Flow Wash Buffer (FWB; RPMI 1640, no phenol red+4% FBS+0.01% sodium-azide). Cells were incubated with the antibody mix for 20 min at room temperature in the dark in 100 µL staining volume in the presence of Super Bright Staining Buffer (eBiosciences), washed twice in FWB, resuspended in 0.5% paraformaldehyde/PBS, and immediately acquired using a Cytek Aurora 5L flow cytometer (Cytek). Antibodies are listed in online supplemental table 1 .
4
Chronic HBV PBMC Characterization
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PBMC samples from the 100 chronic HBV patients were thawed, washed in CTL Anti-Aggregate™ medium (Cellular Technology Limited, CTL) and rested 2h in CTL Test™ medium (Cellular Technology Limited, CTL). Cells were then washed and stained in 96-well plates at 1E6 - 2E6 cells per well with titrated amounts of LIVE/DEAD™ Fixable Blue Dead Cell stain kit (Invitrogen). All surface markers were stained at a final volume of 100µL with titrated amounts of monoclonal antibodies, in the presence of Super Bright Staining Buffer (eBiosciences) for 45 min at room temperature. After the final wash cells were fixed with 1% PFA/PBS and acquired in a BDSymphony® flow cytometer. Analysis was performed with FlowJo v10.6.2. Further information about the antibodies can be found in Supplementary Table 2
5
Multiparametric Flow Cytometry Analysis of Murine Dermal Cells
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Surface staining was performed on dermal cells from ears after enzymatic digestion and processing. To exclude dead cells, cell suspensions were first incubated with LIVE/DEAD Fixable Aqua Dead cell dye (Invitrogen) for 10 min at room temperature. FcγRs were blocked with 2.4G2 anti-mouse CD16/32 antibody (BioXCell) and 0.2% normal rat IgG (BioXCell) for 10 min at 4 °C. For surface staining, cells were stained for 30 min at 4 °C using antibodies: anti-CD45 AF 700 (clone 30-F11), anti-CD45.1 eFlour450 (clone A20), anti-CD45.2 AF 700 (clone 104), anti-Ly6C PerCpCy5.5 (clone HK1.4) (all from eBiosciences); anti-CD11b BV605 (clone M1/70), anti-Ly6G APC (clone 1A8), and anti-CD64 PECy7 (clone X54-5/7.1) (all from Biolegend) in the presence of Brilliant Violet Buffer (BD Biosciences) or Super Bright staining buffer (eBiosciences). Cell events were acquired using the LSRII Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software version 10 (Tree Star).
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