HFSCs and other cell types are isolated as mentioned above and 0.5 million cells are harvested and washed three times with ice cold PBS to remove serum. Cells are resuspended in 100 μL RIPA buffer (50 mM Tris pH8.0, 150 mM NaCl, 1.0% NP40, 0.5% DOC, 0.1% SDS) and lyse with interval vortex on ice for 30 min. After centrifugation, supernatant is transferred into a new tube to detect RA with Mouse Retinoic Acid ELISA kit (Cusabio). ELISA follows instructions on the website of Cusabio (www.cusabio.com ). Minimal light exposure is ensured during whole process.
Mouse retinoic acid elisa kit
Manufactured by Cusabio
The Mouse Retinoic Acid ELISA kit is a laboratory equipment used to detect and quantify the levels of retinoic acid, a signaling molecule, in mouse biological samples. This kit employs the enzyme-linked immunosorbent assay (ELISA) technique to measure the concentration of retinoic acid present in the samples.
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2 protocols using mouse retinoic acid elisa kit
1
Retinoic Acid Quantification from Cells
2
Isolation of Pleural Exudate Cells
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Pleural exudate cells (PLEC) were obtained via washing of the pleural cavity with 2 ml ice-cold PBS followed by 6-8 ml of PBS supplemented with 2 % FCS and 2 mM EDTA using a sterile transfer pipette. Cells were filtered through a sterile pluriStrainer Mini 40 μm cells strainers (pluriSelect) into a 15 ml tube. Worms retained on the filter were removed by back washing with RPMI into a 6 well plate for counting using a dissecting microscope. PBS-only washes were centrifuged, and the supernatant stored in a biobank for future analysis by competitive ELISA for retinoic acid detection using Mouse Retinoic Acid ELISA kit (Cusabio) using manufactures instructions. The cell pellet of the PBS-wash was combined with the cell pellet of the PBS-FCS-EDTA washes and cells were counted using a Nexcelom automated cell counter. For a minority of samples, erythrocytes were lysed prior to cell counting.
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