Facs lyse

Flow cytometric immunophenotyping of leucocytes was performed by analyses of whole blood. The concentration and percentage of CD4 and CD8 T cell populations were measured using a mAb against canine CD4 FITC/CD8 RPE (DC048) purchased from AbD Serotec (Bio-Rad) (UK). Aliquots of 100 μl of whole blood containing approximately 1x10⁶ white blood cells were added to 10 μl of mAbs and incubated for 1 hour in the dark at 4°C. Next, the erythrocytes were lysed, and the leucocytes were fixed by addition of FACSLyse (Becton Dickinson Biosciences, San Josè, CA, USA). After two washes with PBS, the cells were analysed by Cell Quest Pro software on a FACSCalibur flow cytometer (Becton Dickinson Biosciences) equipped with a 488 nm wavelength argon laser and a 635 nm wavelength red diode laser. The results were reported as the percentage of gated cells positive for each cell surface marker.

Samples were obtained at baseline, at 2 h immediately prior to the thrombosis study and at 24 h post exposure, and processed according to previously described protocols [59 (link)]. In brief, blood was taken from an ante-cubital vein using a 21-gauge cannula and anti-coagulated with D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (75 μL; Cambridge Biosciences, UK). Samples were not analysed unless venesection achieved rapid and uninterrupted blood flow. Five minutes after sample collection, samples were stained with the following conjugated monoclonal antibodies: phycoerythrin (PE)-conjugated CD14 (Dako, Denmark), PE-conjugated CD62P, and PE-conjugated CD154 (Becton-Dickinson, UK); PE-conjugated CD40, fluorescein isothiocyanate (FITC)-conjugated CD42a, and FITC-conjugated CD14 (Serotec, USA); and appropriate control isotypes. All antibodies were diluted 1:20. Once stained, samples were incubated for 20 min at room temperature to identify P-selectin and CD40L on the platelet surface and CD40 on the monocyte surface. Platelet–monocyte samples were fixed with FACS-Lyse (Becton-Dickinson). Platelet samples were fixed with 1% paraformaldehyde. Samples were analysed within 24 h using a FACScan flow cytometer (Becton-Dickinson). Platelet–monocyte aggregates were defined as monocytes positive for CD14. Data analysis was performed using FlowJo (Treestar, USA).

Subconfluent HaCaT cells were serum‐starved overnight and incubated overnight at 37°C with LCMV (MOI 0.05 and 0.2). Cells were detached from the 12‐well plate using 1% trypsin and fixed/permeabilized in 500 μl 2 × FACS Lyse (Becton Dickinson, Franklin Lakes, NJ) with 0.05% Tween‐20 for 10 min at room temperature. After washing, intracellular staining was performed for 30 min at room temperature using the LCMV nucleoprotein‐specific antibody VL‐4. After washing, they were resuspended in PBS/1% PFA. Flow cytometric analysis was performed using an LSRII flow cytometer (Becton Dickinson). Raw data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR).

Seventy microlitres of blood were taken with heparinised glass capillaries from the tail. After blocking unspecific binding with 10% mouse serum and Fc receptor blocking antibodies (anti-mouse CD16/32, eBioscience, UK), the cell surface was stained with conjugated antibodies for CD11b (Pe-Cy7; eBioscience, UK), and Ly-6G (IA8 clone, APC-Cy7; BioLegend) for 45 min at 4°C. Red blood cells were lysed using FACS Lyse (BD Bioscience) for 10 min after which cells were washed and resuspended in 200 µl PBS. Cells were analysed on the FACS Canto flow cytometer (BD Bioscience) using FACS Diva software.

By NAVIOS 2 laser 6 colors flow cytometry (Beckman coulter, USA). The acute leukemia panel of fluorescein isothiocyanate (FITC)/phycoerythrin (PE) conjugated monoclonal antibodies (Beckman Coulter, life science, Hielach, USA); IgG1PE, CD45FITC, CD117PE, CD10FITC, CD19PE, HLA‐DR FITC, CD13PE, CD33PE, CD3FITC, CD7PE, C.TDTFITC, C.MPOFITC, CD14PE, CD15FITC, CD34PE, CD61 FITC, GLYCOPHORINPE, and CD56 PE (clone N901) were used for diagnosis and sub‐classification of AML. Schiff's reagent (Merck), erythrocyte lyses solution (FACS lyse BD‐Bioscience), perforating reagent tween 20, and phosphate buffer solution were used to prepare the solutions necessary to carry out the technique [9 ]. Gating was done on the residual normal BM lymphocyte population based on forward and side scatters and their bright expression of CD45. Those gated lymphocytes were analyzed for the percentages of CD3 + (PC5) and the expression of PD1 (VioBright FITC, clone 1.3.1.3) on these CD3+ lymphocytes (Figure 1).