Journal: iScience

Article Title: Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease.

doi: 10.1016/j.isci.2023.106686

Figure Lengend Snippet: Figure 1. Study design and Urinary extracellular vesicles (uEV) quality control (A) Study design for the next generation sequencing of uEV mRNAs to assess urine collections, quality and reproducibility (technical comparisons), origins of uEV transcripts (uEV on Tissue map), and discovery of candidate non-invasive markers for diabetic kidney disease (DKD marker candidates). (B–D) Transmission electron micrographs showing uEV of typical morphology and variable sizes; filamentous structures compatible with Tamm-Horsfall protein filaments are visible in the in-set B1. (E) Western blotting showing the presence of uEV-enriched markers CD9 and PDX in uEV preparations. (F) Representative electropherograms of RNA isolated from uEV and analyzed with bioanalyzer using Agilent Pico kit. An RNA peak between 25 and 200 nt was detected in all samples and with this characteristic shape passed quality control. (G) Examples showing signs of nucleic acid degradation (arrows) in uEV RNA samples and not passing quality control. 24 h (24h); albumin excretion rate (AER); blood pressure (BP); body-mass index (BMI); estimated glomerular filtration rate (eGFR); waist-to-hip ratio (WHR); Genotype-Tissue Expression (GTEx) project; glycated hemoglobin (HbA1C); Lymph Node Carcinoma of the prostate (LnCaP), macroalbuminuria (Macro); microalbuminuria (Micro); non-diabetic control (Control); normoalbuminuria (Normo); messenger RNA sequencing (mRNAseq); Podocalyxin (PDX), type 1 or type 2 diabetes (T1D, T2D); urinary extracellular vesicles (uEV); principal component analysis (PCA); single-nucleus RNA sequencing (snRNA-seq).

Article Snippet: DIREVA samples were collected with PI and centrifuged at 1800g for 10 min at +4 C before freezing as above, and FinnDiane samples were collected either with or completely without these steps. iBEAt samples all contained PI (Complete ULTRA, Mini, EDTA-free, Roche; half tablet per 50 ml urine) and were centrifuged at 3000g for 10 min at RT, after which citrate-EDTA buffer was added (1,0 M citrate, 0,1 M EDTA; 2,5 ml per 45 ml urine) before freezing at -80 C. The uEV were isolated from 30 ml of urine (DIREVA and part of FinnDiane) as above except using a SW-32 rotor (129 168 gmax, k-factor 276, Beckmann-Coulter) and from 8 ml of urine (iBEAt and part of FinnDiane) as described (Barreiro et al.23) using a 70.1 ti rotor (82 656 gmax, k-factor 202, Beckman Coulter, Inc., Brea, CA) for 2 h 30 min at 30,000 rpm at +4 C and no wash. uEV protein and particle quality control Urinary EV samples were analyzed as explained previously.22,66 Briefly, Western blotting of uEVmarker proteins was performed using antibodies against CD9 (SC-13118, Santa Cruz) and podocalyxin (clone 3D3, Novus Biologicals).

Techniques: Control, Next-Generation Sequencing, Marker, Transmission Assay, Western Blot, Isolation, Expressing, RNA Sequencing

Journal: Pakistan Journal of Medical Sciences

Article Title: Differentiation of CD117 + Amniotic Fluid Stem Cells towards Nephron Progenitors

doi: 10.12669/pjms.38.6.4887

Figure Lengend Snippet: Characterization of NPCs colonies for WT1 by immunofluorescence staining as performed on three different amniotic fluid derived NPCs. This figure shows immunofluorescence analysis for positive nuclear marker WT1 (a) Bright field image of NPCs at 40x showed irregular with ill-defined borders having tightly packed cells for the three different amniotic fluid derived NPCs. (b) Counter DAPI nuclear stain showed blue color of Nucleus. (c) Characteristic of NPCs, WT1 is shown in nuclear compartment of cells.

Article Snippet: The amniotic fluid samples were washed thoroughly with Phosphate Buffered Saline then fixed at 1:1 acetone: methanol for twenty minutes at 25°C, after washing with Buffer Saline containing tween 20 (PBST) three times Triton X-100 was use for permeabilization of cell membrane then again washed with PBST, it was clogged by bovine serum albumin (BSA) 1% plus PBS (without Calcium &Magnesium) for 30 minutes at Room Temperature and incubated with primary monoclonal unconjugated antibody WT1 (WT1/857 + 6F-H2 clone, Novus Biologicals, Biotechne, USA) as positive and CD117 (CD117 APC, Thermofisher, 104D2) as a negative control for an hour.

Techniques: Immunofluorescence, Staining, Derivative Assay, Marker

Journal: International Journal of Nanomedicine

Article Title: Evaluation of adipose-derived stem cells for tissue-engineered muscle repair construct-mediated repair of a murine model of volumetric muscle loss injury

doi: 10.2147/ijn.s101955

Figure Lengend Snippet: Figure 1 Characterization of rat adipose-derived stem cells (ADSCs) in culture. Notes: ADSCs were cultured after isolation and cells at passage 2 were either seeded on glass coverslips and cultured in proliferation media in an undifferentiated state for 3 days or processed for FACS analysis. Expression of standard surface markers for ADSCs was confirmed by staining of CD73 (A) and CD90 (B). C and D depict percentage of CD73 and CD90 cells by FACS analysis. Absence of any contaminating muscle progenitor cells in ADSC cultures was revealed by the absence of IF staining for nuclear muscle markers MyoD (E), myogenin (F), and a nonspecific ADSC surface marker Sca I (G). FACS analysis (n=3) estimated that 89.42%±3.2% of cells expressed CD73, whereas CD90 was expressed by 92.80%±2.4% cells (H). FACS analysis also confirmed the negative staining of muscle markers (Pax7, MyoD, and myogenin) and absence of nonspecific surface marker (Sca I) in ADSCs. M1, represents gate used to define the positive cell population. Data is presented as mean ± SD. Abbreviations: FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; DAPI, 4’, 6-diamidino-2-phenylindole.

Article Snippet: Table 1 surface and nuclear marker expression in cultured aDscs and MDcs Marker Source Class Made in 10 dilution ADSCs MDCs surface markers cD73 BD Biosciences Igg1 Mouse 1:10 (Facs); 1:200 (IF) +ve NT cD90 BD Biosciences Igg1 Mouse 1:10 (Facs); 1:200 (IF) +ve NT cD34 Novus Biologicals Igg1 Mouse 1:10 (Facs); 1:200 (IF) -ve NT sca I abcam Igg2a Mouse 1:10 (Facs); 1:200 (IF) -ve NT Nuclear markers Pax7 DhsB Igg1 Mouse 1:10 (Facs); 1:50 (IF) -ve +ve MyoD Dako Denmark a/s Igg1 Mouse 1:10 (Facs); 1:50 (IF) -ve +ve Myogenin (F5D) DhsB Igg1 Mouse 1:10 (Facs); 1:50 (IF) -ve +ve Notes: source details: BD Biosciences (san Jose, ca, Usa); Novus Biologicals (littleton, cO, Usa); abcam (cambridge, UK); DhsB (Iowa city, Ia, Usa); Dako Denmark a/s (glostrup, Denmark).

Techniques: Derivative Assay, Cell Culture, Isolation, Expressing, Staining, Marker, Negative Staining, Fluorescence, FACS