Two hours after transfection, Ang II or PBS was added into medium to pretreat the transfected VSMCs for 24 hours. The cell supernatant or separated serum of mice was collected to determine levels of TNF-α (RAB0477; Sigma-Aldrich Chemical Company, St Louis, MO, USA), COX-2 (ab210574, Abcam Inc., Cambridge, MA, USA), IL-6 (RAB0308; Sigma-Aldrich Chemical Company, St Louis, MO, USA), and IL-1β (RAB0274; Sigma-Aldrich Chemical Company, St Louis, MO, USA) by ELISA. Because NO would immediately transform into NO2- and further transform into NO3-, Griess reagent was used to measure the content of NO (KGE001, R&D Systems, Minneapolis, MN, USA) based on the reaction between nitrite and nitrate. Three replicates were set for each group.
Rab0308
Manufactured by Merck Group
Sourced in United States
RAB0308 is a laboratory centrifuge designed for general purpose applications. It features a maximum speed of 4,000 rpm and a maximum capacity of 6 x 50 mL tubes. The centrifuge is compact, with dimensions of 25 x 35 x 21 cm, and has a weight of approximately 10 kg.
Automatically generated - may contain errors
Lab products found in correlation
Rab0477, by Merck Group (7 mentions)
Rab0274, by Merck Group (4 mentions)
Ab210574, by Abcam (1 mentions)
Kge001, by R&D Systems (1 mentions)
Bms6002, by Thermo Fisher Scientific (1 mentions)
Mak008, by Merck Group (1 mentions)
Rab1089, by Merck Group (1 mentions)
Ab133357, by Abcam (1 mentions)
Ab10558, by Abcam (1 mentions)
13 protocols using rab0308
1
Evaluating Inflammatory Markers in VSMCs
2
Measuring Inflammatory Cytokines in Plasma
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To measure the plasma levels of TNFα, IL-1β and IL-6 blood samples were collected at experiment endpoint. ~200 μl of blood was collected and centrifuged at 5,000 rpm for 10 min at 4°C. The plasma levels of TNFα (RAB0477; Sigma-Aldrich, St. Louis, MO, USA), IL-1β (BMS6002; Thermo Fisher Scientific) and IL-6 (RAB0308; Sigma-Aldrich) were determined via ELISA using the manufacturer’s instructions.
3
Biochemical Evaluation of Wound Healing
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In acute wound study, before euthanizing the mice, blood was collected via retro-orbital plexus for serum separation. The serum amassed from each mice group at day 3, 8 and 14 post-wound was estimated for hydroxyproline (indirect biochemical marker of collagen), IL-6 (interleukin-6, a pro-inflammatory cytokine) and VEGF (a proliferative phase marker) levels. Collagen was quantified using colorimetric based hydroxyproline assay kit (MAK008-Sigma-Aldrich, USA) whereas IL-6 and VEGF levels were determined using ELISA kits (RAB0308 and RAB0509, Sigma-Aldrich, USA) following manufacturer’s protocol.
In case of diabetic study, wound tissue collected at each timeline (3, 10 and 18 days post wound) was homogenized in 0.1 M PBS and then centrifuged at 10000 rpm for 15 min at 4 °C. The obtained skin homogenates were biochemically estimated for hydroxyproline, IL-6, TGF-β (transforming growth factor) and TNF-α (tumor necrosis factor) levels using ELISA (following manufacturer’s protocol). The absorbance was taken at specified wavelength to calculate the amount of these factors at a particular time interval to study the effect of NCs treatment on the wound healing events.
In case of diabetic study, wound tissue collected at each timeline (3, 10 and 18 days post wound) was homogenized in 0.1 M PBS and then centrifuged at 10000 rpm for 15 min at 4 °C. The obtained skin homogenates were biochemically estimated for hydroxyproline, IL-6, TGF-β (transforming growth factor) and TNF-α (tumor necrosis factor) levels using ELISA (following manufacturer’s protocol). The absorbance was taken at specified wavelength to calculate the amount of these factors at a particular time interval to study the effect of NCs treatment on the wound healing events.
4
Cytokine and Organ Function Analysis
5
Endothelial Function Biomarker Assay
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Adenosine (A4036), AMP (01930), ATP (A26209), Evans blue (E2129), HBSS (H9269), LPS (E. coli 055:b5, L2880), and phosphate-buffered saline (PBS) (P5493), as well as the soluble ICAM-1, VCAM-1, and IL-6 serum levels (RAB0220, RAB0506, RAB0308) were purchased from Sigma-Aldrich/Merck (Saint Louis, MO, USA).
6
Quantifying Pro-Inflammatory Cytokines
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7
Astrocyte Response to Radiation
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Human ELISA kits for lysates for TNFa (Sigma; RAB1089), IL6 (Sigma; RAB0308), and IL8 (Sigma; RAB0319) were used according to manufacturer's instructions on lysates from astrocytes derived from 4 individual donors 24 hours after irradiation with 10 Gy. TNFa values were normalized to total protein in lysates. IL6 and IL8 were used as markers of astrocyte activation.
8
Inflammatory Factors Detection and Immune Cell Characterization
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Serum was isolated from blood samples and used to detect in ammatory factors IL-6 (RAB0308, Sigma) and INF-γ (RAB0224, Sigma) using ELISA kits. Mesenteric lymph nodes were xed with 4% paraformaldehyde (0.01 M, pH 7.4, 4°C) for 24-48 hours, and were routinely dehydrated, transparent, waxed, and embedded. Sections with a thickness of 5 µm were sliced on the para n sectioning machine and placed on the slides treated with poly acid for baking at 60°C for 4-6 hours and stored.
Immunohistochemistry for CD45 and CD11b antigens was performed. Antigens were unmasked by microwaving sections in 10 mmol/L citrate buffer, pH 6.0 (15 min), and immunostaining was undertaken using the avidinbiotinylated enzyme complex method with antibodies against CD45 (ab10558, Abcam) at a concentration of 1 µg/ml, CD11b (ab133357, Abcam) at a concentration of 1 µg/ml, and biotinconjugated secondary antibody at a concentration of 1 µg/ml.
Immunohistochemistry for CD45 and CD11b antigens was performed. Antigens were unmasked by microwaving sections in 10 mmol/L citrate buffer, pH 6.0 (15 min), and immunostaining was undertaken using the avidinbiotinylated enzyme complex method with antibodies against CD45 (ab10558, Abcam) at a concentration of 1 µg/ml, CD11b (ab133357, Abcam) at a concentration of 1 µg/ml, and biotinconjugated secondary antibody at a concentration of 1 µg/ml.
9
Quantification of Plasma Inflammatory Markers
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Plasma IL-6, TNF-α, and NfL quantification was determined by ELISA for the dose optimization study and the delayed intervention study. Blood was collected from the submandibular vein into a microtube containing EDTA as an anticoagulant. Samples were centrifuged for 15 min at 1,000 × g at 2–8°C within 30 min of collection. Ten microliters of the plasma supernatant were stored at −20°C until analysis. Plasma was diluted at 1:10 in sterile PBS, then IL-6, TNF-α, and NfL quantification by ELISA was performed in duplicate for each animal (Sigma-Aldrich; ref. RAB0308, Sigma-Aldrich ref. RAB0477, and Novus Biologicals, Toronto, Canada, ref. NBP2-80299, respectively).
10
Cytokine and Nitric Oxide Measurement
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ELISA detection kits were used to determine the levels of TNF-α (cat. no. 88-7340-22 Thermo Fisher Scientific, Inc.), IL-6 (cat. no. RAB0308 Sigma-Aldrich; Merck KGaA) and NO (cat. no. A013-2, Nanjing Jiancheng Bio-Engineering Institute co., Ltd., Nanjing, china), according to the manufacturer's protocol. The absorbance values at 450 and 520 nm were obtained, and the respective contents of TNF-α, IL-6 and NO were confirmed according to the standard curves.
Statistical analysis. The measurement data (mean ± standard deviation) were compared based on t-test, while multigroup comparisons were examined using analysis of variance, followed by Bonferroni's post-hoc test. Statistical analyses were conducted using SPSS 16.0 software (SPSS, Inc., chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Statistical analysis. The measurement data (mean ± standard deviation) were compared based on t-test, while multigroup comparisons were examined using analysis of variance, followed by Bonferroni's post-hoc test. Statistical analyses were conducted using SPSS 16.0 software (SPSS, Inc., chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
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