All blood samples were stained within 1 h after collection or were immediately stored at 4 °C, in accordance with the standardization recommendations for mHLA-DR measurement [18, 19].
Clinical specimens, except for blood samples, were stained with auramine-O-fluorochrome dye and examined microscopically for acid-fast bacilli (AFB).
In the first validation experiment 4 spots from 8 different patient blood samples were stained by IHC for AR expression and presented blindly to two different operators to both enumerate the CTC and score the staining intensity of each cell.
For this purpose, whole blood samples were stained with PE-labelled HLA-A*0201 HLA-A*0201ompentameroImmune Ltd., Oxford, UK), folded around the HER-2/neu369–377-specomplexProImmune
All peripheral blood samples were stained for flow cytometric analysis using CYTO-STAT tetraCHROME monoclonal antibody, followed by analysis of total lymphocyte counts (TLC) and lymphocyte subset counts of CD3+, CD4+, CD8+, CD19+, and CD56+ with a Cytomics FC500 Flow Cytometry System (Beckman Coulter, Inc., Miami, FL, USA) with a 488 NM argon laser.
Furthermore, to characterize T-cell immunoglobulin mucin 3 positive (Tim-3+) or CD38+ T cells, blood samples were stained with the following antibodies: Pcy5-conjugated anti-CD3, FITC-conjugated anti-CD4, FITC-conjugated anti-CD8, PE-conjugated anti-Tim-3 (R&D Systems, Minneapolis, MN, USA) or PE-conjugated anti-CD38 (Beckman Coulter).
The thick and thin blood smears prepared on glass slides at the time of blood sampling were stained with Giemsa stain and examined following standard protocols [ 20].
The thick and thin blood films prepared on glass slide at the time of blood sampling were stained with 10 % Giemsa and examined following standard protocols [ 28].
All non-blood samples were stained with Gram, Ziehl Neelsen and auramine and cultured onto blood and chocolate agar media, MacConkey agar, Brucella agar Lowenstein Jensen and/or Middlebrook medium (BACTEC MGIT 960, Becton Dickinson, Diagnostic Systems, Spark, MD).
Pre- and post-dose cynomolgus monkey whole-blood samples were stained with anti-CD123 (7G3), anti-CD20 (2H7), anti-CD14 (M5E2), and anti-HLADR (L243) (BD Biosciences) to detect pDCs and with A647-labeled 24F4A to detect BDCA2.
In addition, to characterize Tim-3+ and HLA-DR + monocytes, whole-blood samples were stained with antibodies against FITC-conjugated anti-CD14 and allophycocyanin-conjugated anti-Tim-3 (R&D Systems) or PE antibodies against major histocompatibility complex class II cell surface receptor encoded by the human leukocyte antigen (anti-HLA-DR) (BD Biosciences).
