phosphate buffered saline pbs  (Repligen Corp)

Journal: Journal of Extracellular Vesicles

Article Title: Systemic Inflammation Modulates Clearance and Drives Extra‐Hepatic Distribution of Extracellular Vesicles

doi: 10.1002/jev2.70256

Figure Lengend Snippet: I.P. administered EV biodistribution in tissues. (a) Schematic workflow for studying biodistribution of exogenous EVs in vivo. (b–g) EV accumulation in the respective tissues was quantified as % of injected dose—% ID (organ/mouse) or % ID/mL of plasma employing the bioluminescent assay. The numbers display RLU fold change in EV accumulation in LPS‐primed mice over the healthy mice in the brain (b), lungs (c), liver (d), spleen (e), kidney (f) and plasma (g). The acute inflammation was induced by a single dose of LPS at 5 mg/kg 4 h before the EV administration. Based on NTA measurements, EVs were injected I.P. at 1 × 10 11 EVs/dose ( n = 4–5 mice) for 0.5, 2, 4 and 24 h before the blood and tissue collection according to the Methods section for the subsequent analysis. A control group was used for the normalisation of the data ( n = 4). Black—non‐primed healthy mice, pink—LPS‐primed mice. Arrows indicate the fold change in EV accumulation in LPS‐primed mice over healthy mice (primed with PBS). Statistical analysis was performed by two‐way ANOVA. ** Represents p < 0.01, *** p < 0.001 and **** p < 0.0001. The results represent mean ± SD. EV, extracellular vesicle; ID, injected dose; I.P., intraperitoneally; LPS, lipopolysaccharide; NTA, nanoparticle tracking analysis; PBS, phosphate buffered saline; RLU, Relative Luminescent Units.

Article Snippet: EVs from pre‐cleared CM were further subjected to diafiltration with double volume of pre‐filtered phosphate buffered saline (PBS) and concentrated to the volume of about 50 mL by using tangential flow filtration (TFF) with the KrosFlo KR2i TFF System (Repligen, USA) equipped with modified polyethersulfone (mPES) hollow fibre filters with 300 kDa membrane pore size (MidiKros, 370 cm 2 surface area, SpectrumLabs, D06‐E300‐05‐N) as a cutoff membrane at a flow rate of 100 mL/min (transmembrane pressure at 3.0 psi and shear rate at 3700 s −1 ) as described previously (Corso et al. ).

Techniques: In Vivo, Injection, Clinical Proteomics, Control, Saline

Journal: Journal of Extracellular Vesicles

Article Title: Systemic Inflammation Modulates Clearance and Drives Extra‐Hepatic Distribution of Extracellular Vesicles

doi: 10.1002/jev2.70256

Figure Lengend Snippet: EV association with immune and non‐immune cells and biodistribution to various organs. % of EV positive (a) B cells; (b) CD4 T cells; (c) CD4− T cells; (d) myeloid cells; (e) macrophages; (f) neutrophils in the respective tissues: the liver, lungs, spleen, BM and blood; (g) hepatocytes and (h) plasma. Inflammation in mice was induced by a single dose of LPS at the concentration 5 mg/kg I.P. for 4 h before the administration of EVs. EVs were injected I.V. at 2 × 10 11 EVs/dose ( n = 3 mice) for 2 h. A control group (PBS) was used for the normalisation of the data ( n = 1–2). The immune cell panel was changed depending on the cell type analysis. Cells from each organ were stained for the hematopoietic differentiation marker CD45 (CD45+), macrophages (F4/80+), myeloid (GR1+/Cd11b+), B cells (B220), CD4 T cells (CD4+/CD3+), CD4− T cells (CD4−/CD3+), neutrophils (CD11b+/Ly6G+) and evaluated by Flow cytometry. Hepatocytes were gated after excluding immune cells. Cells associated with EVs were mNG positive. The values were normalised to untreated control mice and calculated as % mNG+ cells of viable or mNG+ cell counts/µL of plasma. Black—non‐primed healthy mice, pink—LPS‐primed mice. ND, non‐detectable. Statistical analysis was performed by a two‐tailed t test. *Represents p < 0.05, **represents p < 0.01, *** p < 0.0001 and **** p < 0.0001. The results represent mean±SD. BM, bone marrow; EV, extracellular vesicle; I.P., intraperitoneally; I.V., intravenously; LPS, lipopolysaccharide; mNG, mNeonGreen; PBS, phosphate buffered saline.

Article Snippet: EVs from pre‐cleared CM were further subjected to diafiltration with double volume of pre‐filtered phosphate buffered saline (PBS) and concentrated to the volume of about 50 mL by using tangential flow filtration (TFF) with the KrosFlo KR2i TFF System (Repligen, USA) equipped with modified polyethersulfone (mPES) hollow fibre filters with 300 kDa membrane pore size (MidiKros, 370 cm 2 surface area, SpectrumLabs, D06‐E300‐05‐N) as a cutoff membrane at a flow rate of 100 mL/min (transmembrane pressure at 3.0 psi and shear rate at 3700 s −1 ) as described previously (Corso et al. ).

Techniques: Clinical Proteomics, Concentration Assay, Injection, Control, Staining, Marker, Flow Cytometry, Two Tailed Test, Saline