Mouse il 3 quantikine elisa kit

Lysates of whole fetal spleen or liver tissues were prepared using the Qproteome Mammalian Protein Prep Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Hematopoietic cells (CD45+/CD45+Ter119+/Ter119+), endothelial cells (CD45−Ter119−CD31+LYVE-1−), unclassified cells (CD45−Ter119−CD31−LYVE-1−) and CD51+ cells among unclassified cells (CD45−Ter119−CD31−LYVE-1−CD51+) were sorted. Lysates were prepared using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL) and a protease inhibitor from the Qproteome Mammalian Protein Prep Kit (Qiagen). Samples were centrifuged at 12,000 rpm for 10 minutes at 4°C. Supernatants containing soluble protein were collected and protein concentration was quantitated using the Bradford reagent (Bio Rad, Hercules, CA) according to the manufacturer's instruction. The optical density (O.D.) at 540 nm was measured using a Thermo Multiskan FC plate reader (Thermo Fisher Scientific). SCF, IGF-1, IL-3 and EPO ELISA assays were conducted using a mouse SCF Quantikine ELISA kit (R&D Systems, Minneapolis, MN), a mouse IGF-1 Quantikine ELISA kit (R&D Systems), a mouse IL-3 Quantikine ELISA kit (R&D Systems) and a mouse EPO Quantikine ELISA kit (R&D Systems), according to the manufacturer's instructions. O.D.s at 450 nm and 540 nm were measured using a Thermo Multiskan FC plate reader.

Expression of IL-3, IL-5, IL-13, and GM-CSF cytokines was measured on CD4⁺ T cell supernatants with the Mouse IL-3 Quantikine ELISA Kit (R&D Systems), LEGEND MAX Mouse GM-CSF ELISA Kit, Mouse IL-5 ELISA MAX Deluxe (BioLegend), and Invitrogen IL-13 Mouse ELISA Kit (Thermo Fisher Scientific), following the manufacturers’ instructions. TSLP expression was measured in mouse plasma and breast tumors/mammary glands using the LEGEND MAX Mouse TSLP ELISA Kit (BioLegend). Expression of TSLP in human breast tumor and healthy tissue was measured using the LEGEND MAX Human TSLP ELISA Kit (BioLegend) following manufacturer’s instructions. The same amount of total protein, quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), was used for the assays. Optical densities were measured on a Synergy Neo2 (BioTek) at 450 nm, and cytokine concentrations were calculated with a five-parameter logistic curve using Gen5 Microplate Reader and Imager Software (BioTek).