Evaluation of the functional properties of cryopreserved buffy coat–derived monocytes for monocyte monolayer assay

Abstract

Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies.

Red cell supernatant effects on endothelial cell function and innate immune activation is influenced by donor age and sex

“…Similarly, recent studies show an increased secretion of IL‐8 from monocytes co‐cultured with fresh (≤7 days) supernatants whole blood filtered (WBF) RCC products while transfusion fresh WBF RCCs were associated with higher in‐hospital mortality compared with mid‐aged BC RCC products . Cytokines are low molecular mass glycoproteins that participate in inflammation and immunity against infectious agents as well as the regulation of various biologic systems . They have numerous pro‐ or anti‐inflammatory functions .…”

“…PBMCs were isolated from three buffy coat (BC) components, pooled, cryopreserved in 20% dimethyl sulfoxide (DMSO, Sigma‐Aldrich, Oakville, ON, Canada) and stored in liquid nitrogen (−196°C) as previously described . PBMCs had been stored for 1 year (−196°C) at the time of use in experiments.…”

“…PBMCs had been stored for 1 year (−196°C) at the time of use in experiments. Monocyte monolayers from pooled cryopreserved BC PBMCs for monocyte monolayer assay (MMA) were prepared as previously described . Monocytes were treated with 1 ml of day 7, day 21 or day 42 RCC supernatants (20% v/v in culture media) followed by 4‐h incubation (37°C, 5% CO 2 ).…”

“…Monocytes incubated with 1 ml culture media served as negative controls. Monocyte cell culture supernatants were collected prepared for cytokine analysis as previously described .…”

“…Luminex technology using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panels (EMD Millipore, Toronto, ON, Canada) was used to assess the concentration of 10 cytokines/chemokines secreted into monocyte and HUVECs culture supernatants as previously described . The cytokines/chemokine panel included granulocyte‐monocyte‐colony‐stimulating factor (GM‐CSF), interferon‐gamma (IFN‐γ), interleukin (IL)‐1‐beta (IL‐1β), IL‐4, IL‐6, IL‐8, IL‐10, IL‐12p70, tumour necrosis factor‐alpha (TNF‐α) and monocyte chemo‐attractant protein ‐1 (MCP‐1).…”