Edta tube

Blood was collected in EDTA tubes (Greiner Bio-One, Kremsmünster, Austria), cooled at 4 °C and centrifuged; plasma samples were stored at − 80 °C at the time of IE diagnosis, for subsequent use.
Plasma concentration of pro-ADM (cut-off for positivity 0.38 nmol/L), copeptin (cut-off for positivity 3.9 pmol/L) and PCT (cut-off for positivity 0.064 μg/L) were measured using an automated immunofluorescent assay on a Kryptor system (B.R.A.H.M.S. AG, Henningsdorf, Germany).
Other laboratory parameters were obtained by routine methods used in our Hospital central laboratory, including C-reactive protein (cut-off for positivity 0.3 mg/dL).

Plasma samples were collected for each patient before treatment initiation, after 2 weeks and 4 weeks of treatment, then every 4 weeks until progression, death or unacceptable toxicity. The samples were collected in EDTA tubes (Greiner Bio-One), centrifuged at 2000 g for 10 min, decanted and frozen at −80° C within 4 hours after collection.

Plasma samples collected before treatment initiation of stage IV or unresectable stage III melanoma patients at Nantes University Hospital between January 2014 and March 2017 were included in this study. Plasma samples were collected in EDTA tubes (Greiner Bio-One, Les Ulis, France), centrifuged at 2000× g for 10 min, and frozen at −80 °C within 4 h after venepuncture.
During this time frame, detection of BRAF and NRAS mutation in the patient’s tumour was performed using a combination of allele-specific amplification and Sanger sequencing [26 (link),27 (link),28 (link)]. Our laboratory is accredited in accordance with the International Standard ISO15189.
Patients were treated either by immunotherapy (nivolumab monotherapy or nivolumab–ipilimumab combination) or by targeted therapy for BRAF-mutated patients (vemurafenib alone or vemurafenib + cobimetinib or dabrafenib + trametinib).

Morning fasted blood samples were obtained from the antecubital vein for the analyses of hemoglobin, ferritin, and serum total 25-hydroxyvitamin D, e.g., serum 25(OH)D. Blood for the hemoglobin analysis was drawn into EDTA tubes (Greiner-Bio-One GmbH, Kremsmünster, Austria) and immediately further analyzed with Sysmex XP300 analyzer (SysmexCo., Kobe, Japan). For serum ferritin, the blood was drawn into Vacuette gel serum tubes (Greiner-Bio-One GmbH, Kremsmünster, Austria) and centrifuged for 10 min with 3,600 rpm to collect serum, which was then frozen to −20°C for further analysis. The samples were analyzed with Siemens Immulite 2000 XPI analyzer (Siemens Healthcare Lianberis, United Kingdom), where the serum ferritin was determined by using immunometric chemiluminescence method. The sensitivity of the assay for ferritin was 0.4 µ/L and the precision (CV%) for the assay was 4.6%. The measurements of serum 25(OH)D were performed using electrochemiluminescence immunoassays (ECLIA).

In rabbit, plasma levels of rbBB7 were determined by enzyme-linked immunosorbent assay (ELISA) immunoreactivity to purified recombinant human TG2 (rhTG2). Ear vein bleeds were collected in EDTA tubes (Greiner Bio-One), and plasma prepared by centrifugation at 1000 x g for 15 min at 4°C. ELISA plates were coated with rhTG2 (50 ng/well; Zedira). Wells were washed in PBS/Tween (0.1% v/v), blocked with PBS/Tween 3% BSA (w/v) for 2 h at RT, washed, then plasma (diluted in PBS/Tween 1% BSA) was added and the plates incubated for 1 h at RT. Wells were washed and then incubated with anti-rabbit HRP 1:5000 in PBS/Tween 1% BSA for 1 h at RT and revealed with TMB substrate (Thermo Fisher Scientific). The reaction was stopped with 2 M H₂SO₄, and optical density determined by spectrophotometer at 450 nm. Negative control plasma from rabbits injected with vehicle alone did not exhibit detectable reactivity against rhTG2 in this assay.
In cynomolgus monkeys, zampilimab was measured in lithium-heparinized plasma samples by liquid chromatography–electrospray ionization–tandem mass spectrometry assay using zampilimab (isotopically labeled peptide H₂N– GLPSSIEK–COOH) as internal standard for zampilimab. The method has a limit of quantification of 1 μg/mL for zampilimab.

To detect intracellular cytokines and chemokines by flow cytometry, mice administered with Proleukin^®/IL-2 and saline-treated control groups of the same experiment were injected with 0.25 mg of BFA (Sigma-Aldrich, USA) at the endpoint of the experiment (144 h) and sacrificed 6 h later. Submandibular blood collection was carried out in EDTA tubes (Greiner Bio-One, Austria), and red blood cells (RBCs) were lysed using RBC lysis buffer (Life Technologies, USA) prior to flow cytometry staining. Spleen and lymph nodes were digested in a mixture of collagenase IV (GIBCO, UK), DNase I (Sigma Aldrich, USA) and meshed through a 70 µm filter (Thermo Fisher Scientific, USA) in DMEM medium (Thermo Fisher Scientific, USA). When necessary, cell suspensions were subjected to red blood cell lysis (GIBCO, UK). The single-cell suspension was washed and re-suspended in media supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA).