Serum levels of proteases were measured in blood drawn 4 hrs (unless other specified) after challenge. MMCP1 was evaluated with a commercial ELISA kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions. mcpt7 was evaluated by an ELISA developed in our lab. ELISA plate wells were coated overnight with 0.2 mg/ml of a goat anti-mouse mcpt7 polyclonal Ab (clone AF1937, R&D Systems, Minneapolis, MN). Serial dilutions of serum samples or recombinant standard (R&D Systems, Minneapolis, MN) were loaded into wells and bound mcpt7 was detected with 0.2 mg/ml of a biotin-labeled goat anti-mouse mcpt7 polyclonal Ab (BAF1937, R&D Systems, Minneapolis, MN), followed by streptavidin peroxidase conjugate (Thermo Fisher Scientific, Waltham, MA) and SuperSignal™ ELISA Femto Substrate (Pierce, Waltham, MA) according to the manufacturer’s instructions. Although both assays show the greatest effects of desensitization when serum is obtained within 30 min of challenge, they still show marked effects when serum is obtained up to 4 hrs after challenge. Obtaining serum at the latter time point was frequently necessary to simultaneously test for cytokine responses and because it is difficult to obtain serum when mice are recovering from shock.
Recombinant standard
Manufactured by R&D Systems
Sourced in United States
Recombinant standard is a laboratory product that serves as a reference material for quantitative analysis. It is produced through recombinant DNA technology and can be used to establish standard curves and validate experimental procedures.
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Lab products found in correlation
5 protocols using recombinant standard
1
Measuring Serum Protease Levels
2
Quantitative Measurement of Met, Axl, Egfr, and Akt Kinase Activities
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For detecting Met pan-tyosine phosphorylation, sandwich ELISA-based PathScan kit (CST) was used according to manufacturer’s instructions. For scalable detection of kinase activities of Met, Axl, Egfr, and Akt, the following kits were used following manufacturer’s instructions: luminescence-based MET and AXL kinase enzyme systems (Promega) and solid phase sandwich ELISA-based PathScan kits for EGFR and AKT1. For total AXL or AXL phosphorylation measurement, we performed a multiplexed ELISA using individually identifiable beads (Bio-Rad) as described previously58 (link). Capture antibody for AXL (R&D Systems) and biotinylated anti-phosphotyrosine antibody 4G10 (Millipore Corp.) were used. AXL receptor abundance was quantified by comparison with a recombinant standard (R&D Systems).
3
Cytokine Quantification in TB-Infected Macrophages
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Collected cell culture supernatants from the M. tb-infected THP-1 macrophages or MDMs were passed through a 0.2 μM filter to remove any bacteria. Supernatants were assayed for cytokines by a sandwich ELISA, which was conducted following the manufacturer's instructions with absorbance recorded at 405 nm on SoftMax Pro ELISA analysis software (Molecular Devices). The IFN-γ, IL-1β, and TNF-α in the culture supernatants were quantified by comparison with the appropriate recombinant standard (purchased from R&D Systems).
4
VEGF Quantification in Wound Fluid
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The amount of VEGF in wound fluid was measured using enzyme-linked immunosorbent assay using available reagents and recombinant standards (R&D Systems, Minneapolis, USA) according to manufacturer's instruction only on 5th-day samples. The VEGF assay has a minimum sensitivity of 30 pg/ml.
The concentration of total protein (TP) in wound fluid was determined by the Lowry's method using the commercial kit (Pars Azmoon, Iran).
The VEGF concentration of wound fluid was reported after correction by the TP level (VEGF/TP × 107).
The concentration of total protein (TP) in wound fluid was determined by the Lowry's method using the commercial kit (Pars Azmoon, Iran).
The VEGF concentration of wound fluid was reported after correction by the TP level (VEGF/TP × 107).
5
Quantification of Plasma VEGF and sFlt-1
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Plasma VEGF levels were determined in duplicates using ELISA kit from Abnova (Walnut, CA) with the sensitivity of 5 pg/ml. Circulating levels of sFlt-1 in plasma were measured using commercially available reagents and recombinant standards (R&D Systems, Minneapolis, MN, USA). All samples were assayed in duplicate. Standards and control samples were run simultaneously for validation. The minimum detection limit for sFlt-1 was 3.5 pg/ml. Inter- and intra-assay coefficient of variations for the assays were <10%. The assay kit measures total plasma sFlt-1.
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