DataSheet1_A deep 96-well plate RBC storage platform for high-throughput screening of novel storage solutions.PDF

Background: Red blood cell (RBC) storage solutions, also known as additive solutions (ASs), first developed in the 1970s, enable extended storage of RBCs. Unfortunately, the advancements in this field have been limited, due to labor intensive and time-consuming serial in vitro and in vivo testing, coupled with very high commercialization hurdles. This study examines the utility of deep 96-well plates for preliminary screenings of novel ASs through comparison of RBC storage with the standard PVC bags in terms of hemolysis and ATP levels, under both normoxic (N) and hypoxic/hypocapnic (H) storage conditions. The necessity for the presence of DEHP, normally provided by PVC bags, is also examined.Materials and methods: A pool of 2 ABO compatible RBC units was split between a bag and a plate. Each plate well contained either 1, 2 or 0 PVC strips cut from standard storage bags to supply DEHP. The H bags and plates were processed in an anaerobic glovebox and stored in O2 barrier bags. Hemolysis and ATP were measured bi-weekly using standard methods.Results: Final ATP and hemolysis values for the plate-stored RBCs were comparable to the typical values observed for 6-week storage of leukoreduced AS-3 RBCs in PVC bags under both N and H conditions. Hemolysis was below FDA and EU benchmarks of 1% and 0.8%, respectively, and excluding DEHP from plates during storage, resulted in an inconsequential increase when compared to bag samples.Discussion: In combination with high-throughput metabolomics workflow, this platform provides a highly efficient preliminary screening platform to accelerate the initial testing and consequent development of novel RBC ASs.</p

Differentially expressed protein spots in AQP4^−/− quadriceps muscle compared to WT.

<p>Only protein spots that were present on every gel (n = 4 separate comparisons) and demonstrating changes with significance p<0.05 were accepted as being differentially expressed. The proteins were identified by ESI-IT and accession numbers are given.</p

Protein-protein interaction map of the MS-identified proteins.

<p>Protein IDs obtained upon MS-based identification of spots of interest were uploaded to String 8.3 for protein-protein interaction mapping. High degree nodes (GAPDH and VCP) and three main clusters of proteins (right, bottom-left, upper-left) were individuated.</p

Sarcolemma integrity assay.

<p>To examine infiltration of blood serum proteins into damaged muscle fibers, WT, AQP4^−/− and mdx mice were intraperitoneally injected with Evans blue dye. M<i>dx</i> quadriceps displays many Evans blue dye–positive fibers (visualized by red fluorescence), due to membrane damage. Evans blue dye was minimally detected in muscle from WT and AQP4^−/− mice, demonstrating that the absence of AQP4 expression did not perturb sarcolemmal integrity. Scale bar: 50 µm.</p

Empty vector (false positive).

<p>Empty vector (false positive).</p

Interactome of the NBN full-length protein.

<p>The interactome was gleaned from SDS-PAGE-MS/MS analysis of the streptavidin chromatography eluates obtained from cells expressing the full-length NBN protein (<b>A</b>) in control conditions or (<b>B</b>) following 2 Gy of X-rays. Colored nodes indicate those proteins that have been identified experimentally in the present study, while grey nodes indicate likely additional interactors that are predicted on the basis of evidences available from the literature. For details, see the text.</p

Interactome of the p70 fragment.

<p>The interactome was gleaned by merging the results obtained through SDS-PAGE-MS/MS analysis of the streptavidin chromatography eluates obtained from cells expressing the p70 protein fragment (<b>A</b>) in control conditions or (<b>B</b>) following 2 Gy of X-rays. For details, see the text.</p

p70 fragment interactors.

<p>p70 fragment interactors.</p

(A) Measurement of NAD⁺ levels in HEK293 transfected cells.

<p>Cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5, 2, and 24 h. The concentration of NAD⁺ was determined using 2×10⁵ transfected cells. Error bars represent the standard deviation from two independent experiments. (**p<0.01; *p<0.05). (<b>B</b>) <b>DSBs repair analysis evaluated by γ-H2AX foci.</b> HEK293 cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5, 2, and 24 h. Fixed cells were stained with anti-γ-H2AX, and the number of γ-H2AX foci was counted in 50 cells/experiment, in two repeated experiments. (**p<0.01; *p<0.05). (C) Co-immunoprecipitation experiments aimed at evaluating the interaction of MRE11 with the Strep-tagged proteins and PARP1. HEK293 cells transfected with either the full length or the p26 NBN fragment were exposed to 2 Gy of X-rays and harvested after 0.5 h. Protein eluates were immunoprecipitated with MRE11 and blotted with anti-Strep-tag and anti-PARP1 mouse monoclonal antibodies.</p

Evaluation of the Strep-tag recombinant proteins expression and Western blot analysis of NBN, p26 and p70 interactors.

<p>(<b>A</b>) Protein lysates, obtained after 24 and 48 h from HEK293 transient tranfections, were analyzed by Western blot using a StrepMAB-Classic horse radish peroxidase conjugated antibody. The highest level of expression was observed after 48 h from transfection, for all the recombinant proteins. Protein extracts obtained from HEK293 cells transfected with the empty vector were used as negative control. (<b>B</b>) Check of the expression of the Strep-tag recombinant proteins. Immunoblots were performed using the protein eluates obtained from HEK293 transfected cells either untreated or exposed to 2 Gy of X-rays, lysed after 30 minutes and purified by Strep-tag chromatography. Filters were probed with the anti-NBN antibody directed against the full-length protein. (<b>C</b>) Protein eluates obtained from HEK293 transfected cells exposed to 2 Gy of X-rays were lysed after 30 minutes, purified by Strep-tag chromatography, and analysed by Western blot using the following antibodies: anti-53BP1 rabbit polyclonal, anti-ATM mouse monoclonal, anti-ATM pSer1981 mouse monoclonal, anti-BRCA1 mouse monoclonal, anti-CHK2 pThr68 rabbit polyclonal, anti-CHK2 mouse monoclonal, anti-CtIP rabbit polyclonal, γ-H2AX rabbit polyclonal, anti-Hsp90 rabbit polyclonal, anti-MRE11 mouse monoclonal, anti-NBN mouse monoclonal, anti-PARP1 mouse monoclonal, anti-PP2A rabbit polyclonal, anti-PML rabbit polyclonal, anti-SMC1 rabbit polyclonal.</p