1H NMR spectroscopy was used to investigate the metabolic consequences of general anesthesia in the plasma of two groups of patients with diagnosis for non-metastatic colorectal cancer and metastatic colorectal cancer with liver-metastasis, respectively. Patients were treated with etomidate or propofol, two frequently used sedation agents. Plasma samples were obtained via Ficoll separation. Here, we demonstrated that this procedure introduces a number of limitations for NMR-based metabolomics studies, due to the appearance of spurious signals. Nevertheless, the comparison of the 1H NMR metabolomic profiles of patients treated with etomidate or propofol at equipotent dose ranges was still feasible and proved that both agents significantly decrease the plasma levels of several NMR-detectable metabolites. Consequently, samples collected during anesthesia are not suitable for metabolic profiling studies aimed at patient stratification, because interpersonal variability are reduced by the overall depression of metabolites levels. On the other hand, this study showed that plasma metabolomics could represent a valuable tool to monitor the effect of different sedation agents and/or the individual metabolic response to anesthesia, providing hints for an appropriate tuning of personalized sedation procedures. In our reference groups, the metabolomic signatures were slightly different in patients anesthetized with etomidate versus propofol. The importance of standardized collection procedures and availability of exhaustive metadata of the experimental design for the accurate evaluation of the significance of the observed changes in metabolites levels are critically discussed.

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

For immune monitoring studies during HIV vaccine clinical trials, whole blood specimens from HIV seropositive (HIV+) patients may be collected at multiple sites and sent to a central location for peripheral blood mononuclear cell (PBMC) isolation, cryopreservation and functional evaluation. In this study we show a comparison of two PBMC preparation options, Ficoll density gradient separation (Ficoll) and Cell Preparation Tubes (CPT) using shipped whole blood specimens from 19 HIV+ patients (CD4 > 350, viral load < 50). The pre- and post- cryopreservation performance of samples collected by these two methods were compared by assessment of antigen-specific IFNgamma expression in CD8+ and CD8- T cells, cellular viability, and cellular recovery.

Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasm's volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders also affect the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic H57 but Ficoll crowders increase substrate binding more than PEG molecules.

Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.

G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic.

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral:7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.

The analysis of circulating tumor cells (CTCs) is important for cancer diagnosis and prognosis. Microfluidics has been employed for CTC analysis due to its scaling advantages and high performance. However, pre-analytical methods for CTC sample preparation are often combined with microfluidic platforms because a large sample volume is required to detect extremely rare CTCs. Among pre-analytical methods, Ficoll-Paque™, OncoQuick™, and RosetteSep™ are commonly used to separate cells of interest. To compare their performance, we spiked L3.6pl pancreatic cancer cells into healthy blood samples and then employed each technique to prepare blood samples, followed by using a microfluidic platform to capture and detect L3.6pl cells. We found these three methods have similar performance, though the slight edge of RosetteSep™ over Ficoll-Paque™ is statistically significant. We also studied the effects of the tumor cell concentrations on the performance of the frequently used Ficoll-Paque™ method. Furthermore, we examined the repeatability and variability of each pre-analytical technique and the microfluidics-enabled detection. This study will provide researchers and clinicians with comparative data that can influence the choice of sample preparation method, help estimate CTC loss in each pre-analytical method, and correlate the results of clinical studies that employ different techniques.

The five members of the family of tumor suppressors ING contain a Plant Homeodomain (PHD) that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3) with an affinity in the low micromolar range. Here, we use NMR to show that in the presence of 15% Ficoll 70, an inert macromolecular crowding agent, the mode of binding does not change but the affinity increases by one order of magnitude. The affinity increases also for unmethylated histone H3 tail, but the difference with H3K4me3 is larger in the presence of Ficoll. These results indicate that in the cellular milieu, the affinity of the ING proteins for their chromatin target is larger than previously thought.

The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFβ1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease Vmax and Km for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in Vmax is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD+ product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this "tuning" of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.

We have compared three monocyte isolation procedures for their suitability in the analysis of cytokine mRNA expression in circulating monocytes. Monocytes were isolated from peripheral blood (1) using antiCD14 coated magnetic beads, (2) by Ficoll centrifugation, or (3) by Ficoll centrifugation followed by plastic adherence. The effect of the isolation procedure on the cytokine mRNA expression levels of the isolated monocytes was evaluated by RT-PCR. Results show that the expression of cytokine mRNAs determined in monocytes isolated using antiCD14 coated magnetized beads reflects best the cytokine mRNA levels in circulating monocytes. We subsequently applied this method to the analysis of cytokine mRNA expression levels in rheumatoid arthritis and control monocytes, which revealed that RA and control monocytes isolated by antiCD14 beads produce similar, very low TNF alpha, IL-1beta, and IL-8 mRNA levels.

Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.

High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.

We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.

The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase--a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.