Early Immunological Effects of Ischemia-Reperfusion Injury: No Modulation by Ischemic Preconditioning in a Randomised Crossover Trial in Healthy Humans

Ischemic preconditioning (IPC) has been protective against ischemia-reperfusion injury (IRI), but the underlying mechanism is poorly understood. We examined whether IPC modulates the early inflammatory response after IRI. Nineteen healthy males participated in a randomised crossover trial with and without IPC before IRI. IPC and IRI were performed by cuff inflation on the forearm. IPC consisted of four cycles of five minutes followed by five minutes of reperfusion. IRI consisted of twenty minutes followed by 15 min of reperfusion. Blood was collected at baseline, 0 min, 85 min and 24 h after IRI. Circulating monocytes, T-cells subsets and dendritic cells together with intracellular activation markers were quantified by flow cytometry. Luminex measured a panel of inflammation-related cytokines in plasma. IRI resulted in dynamic regulations of the measured immune cells and their intracellular activation markers, however IPC did not significantly alter these patterns. Neither IRI nor the IPC protocol significantly affected the levels of inflammatory-related cytokines. In healthy volunteers, it was not possible to detect an effect of the investigated IPC-protocol on early IRI-induced inflammatory responses. This study indicates that protective effects of IPC on IRI is not explained by direct modulation of early inflammatory events

Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry.

Live peripheral blood mononuclear cells (PBMCs) can be frozen and thawed for later analyses by adding and removing a cryoprotectant, such as dimethyl sulfoxide (DMSO). Laboratories across the world use various procedures, but published evidence of optimal thawing procedures is scarce.PBMCs were separated from blood collected from healthy Danish blood donors, and stored at -80°C after adding of DMSO. The essential steps in the thawing procedure were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable PMBCs.The best-performing washing medium was Roswell Park Memorial Institute (RPMI) 1640 at 37°C with 20% fetal bovine serum. When using 10 mL washing medium in a 15-mL Falcon tube, samples should be centrifuged for at least 10 minutes at 500 g. We failed to detect any differences between the tested methods of mixing PBMCs with washing medium. Likewise, neither the thawing duration nor centrifugation temperature (20°C and 37°C) had any effect. PBMCs could be incubated (rested) for up to eight hours in a 37°C 5% CO2 incubator without affecting cell counts, but incubating PBMCs for 16 hours significantly decreased viability and recovery. In general, high viability was not necessarily associated with high recovery.Changing the thawing procedure significantly impacted PBMC viability and live cell recovery. Evaluating both viability and live PBMC recovery are necessary to evaluate method performance. Investigation of differential loss of PBMC subtypes and phenotypic changes during thawing and incubation requires further evaluation

Real-Time Relative qPCR without Reference to Control Samples and Estimation of Run-Specific PCR Parameters from Run-Internal Mini-Standard Curves

Background: Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples. Methodology/Principal Findings: In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the ‘‘comparative Ct method’’. Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy. Conclusions/Significance: The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, cop

Impact of washing-medium temperature on viability (A) and PBMC recovery (B).

<p>20 donors included. Ref.: reference group. **: p<0.01.</p

Gating strategy to identify live/dead CD45+ leukocytes.

<p>A) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of dead cells. B) Doublet exclusion C) Identification of CD45 positive cells D) Dead cells identified as 7-AAD positive events. Compensation for spectral overlap was not required.</p

Comparing two ways of mixing PBMCs and washing medium by viability (A) and absolute count of live PBMCs (B).

<p>Paired samples 20 donors included. Ref.: reference group. NS: non-significant. **: p<0.01.</p

Centrifugation time and force and PBMC viability (A) and absolute live PBMC count (B).

<p>Paired samples from 18 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.</p

PBMC viability (A) and absolute recovery (B) as a result of varying thawing time in 37°C heated water bath.

<p>Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.</p