The levels of CCL5 and TGF-β in vitro and the levels of Granzyme B, perforin, TGF-β and IL-10 in vivo were detected by ELISA. Kits for CCL5 and Granzyme B were purchased from R&D Systems (CCL5, ELH-RANTES-1; Granzyme B, ELM-GranzymeB-1). Kits for perforin, TGF-β, and IL-10 were purchased from ABclonal (Perforin, RK03133; IL-10, RK00016; TGF-β, RK00055 and RK00057). Cell culture supernatants or tumor tissues were collected and assayed according to the manufacturer’s instructions.
Rk00016
Manufactured by ABclonal
Sourced in China
RK00016 is a high-performance DNA extraction kit designed for the rapid and efficient isolation of DNA from a variety of sample types. The kit utilizes a proprietary silica-based membrane technology to capture and purify DNA, providing a simple and streamlined workflow.
Automatically generated - may contain errors
3 protocols using rk00016
1
Quantifying Immune Markers in Vitro and In Vivo
2
Cytokine Levels in Serum and Liver
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3
Quantification of Cytokine Levels in Mouse Serum
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For the quantitative determination of cytokine levels in serum, the blood of mice was collected from the orbital veins, then serum was separated at room temperature and stored at -80°C. The concentrations of interleukin-2 (IL-2) (pg/mL) (RK00007, ABclonal, China), IL-4 (pg/mL) (RK00036, ABclonal, China), and IL-10 (pg/mL) (RK00016, ABclonal, China) were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits.
Binding ELISA was conducted to verify the affinity of antibody in the supernatant from pVAX-α-PD-1-transfected cells. The procedure carried out was as follows: 96-well plates were pre-coated with 10 μg/mL of PD-1 protein (Sigma) overnight at 4°C. They were subsequently washed with 0.1% Tween 20/PBS and then blocked with 5% bovine serum albumin (BSA) in a humid chamber for 1 hour at room temperature. After blocking, plates were washed three times and incubated at room temperature with supernatant from cells for 1 hour. Next, plates were washed and incubated with HRP goat anti-mouse lgG (H+L) secondary antibody at a dilution of 1:10,000 for 1 hour. The optical density was measured at 450 nm with an automatic ELISA reader (Synergy H1 Microplate Reader, BioTek, USA).
Binding ELISA was conducted to verify the affinity of antibody in the supernatant from pVAX-α-PD-1-transfected cells. The procedure carried out was as follows: 96-well plates were pre-coated with 10 μg/mL of PD-1 protein (Sigma) overnight at 4°C. They were subsequently washed with 0.1% Tween 20/PBS and then blocked with 5% bovine serum albumin (BSA) in a humid chamber for 1 hour at room temperature. After blocking, plates were washed three times and incubated at room temperature with supernatant from cells for 1 hour. Next, plates were washed and incubated with HRP goat anti-mouse lgG (H+L) secondary antibody at a dilution of 1:10,000 for 1 hour. The optical density was measured at 450 nm with an automatic ELISA reader (Synergy H1 Microplate Reader, BioTek, USA).
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