32 multiplex mouse cytokine chemokine magnetic bead panel

Serpinb3a^−/− or Serpinb3a^+/+ Balb/c background bone marrow stromal cells from cell lines irradiated in vitro were collected at 0, 1, 2, 3 or 24 h after 10 Gy irradiation. Fresh bone marrow and the (small intestine) ileum was harvested from Serpinb3a^−/− and Serpinb3a^+/+ Balb/c background strain at days 0, 1, 3 or 5 after 8 Gy TBI and immediately frozen. The cells or tissues were thawed, homogenized in0.1% Tween™ 80 in phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA) and protein quantitated using Bio-Rad Protein Assay (Hercules, CA). The ileum was thawed, and a 4 mg piece was homogenized in 1 ml of 0.1% Tween 80 in PBS and protein quantitated using Bio-Rad Protein Assay (9 (link)). A 32 Multiplex Mouse Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore, Billerica, MA) was used to measure protein concentrations for eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and VEGF. TGF-β concentrations were measured using a TGFBMAG-64K-01kit (EMD Millipore). A MAGPIX® instrument (Millipore Sigma, Burlington, MA) was used to run the assays (9 (link)).

A list of the 33 proteins assayed and known primary function is shown in Table 1. A Biorad protein assay was run for each tissue type (plasma and intestine) and experimental groups (For example, 19.75 Gy WAI, 19.75 Gy WAI + LR, 19.75 Gy WAI + IL-22, and 19.75 Gy WAI + LR-IL-22) using a Biotek Epoch microplate-spectrophotometer (Biotek Instruments, Inc., Winooski, VT). We utilized a TGF-β1 Single Plex Magnetic Bead Kit, as well as a 32 Multiplex Mouse Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore, Billerica, MA, USA) that tested protein concentrations for each protein (Milliplex® for Luminex® xMAP® Technology). Each kit (MCYTOMAG-70K, TGFBMAG-64K-01) provided reagents for use in the Luminex Protein Immunoassay, which included 1 vial of Mouse Cytokine/Chemokine Standard, 2 vials Mouse Cytokine/Chemokine Quality Controls, 1 vial Serum Matrix, 96-Well Plate, 30 mL Assay Buffer, 60 mL 10X Wash Buffer, 3.2 mL bottle Mouse Cytokine Detection Antibodies, 1 3.2 mL bottle Streptavidin-Phycoerythrin. The 32 Multiplex kit (MCYTOMAG-70K) provided 1 3.5 mL pre-Mixed 32-plex Beads. The TGF-β1 kit (TGFBMAG-64K-01) provided the above reagents, as well as 5 mL Sample Diluent, 1.0 mL 1.0 N HCl, 1.0 mL 1 N NaOH, and 3.5 mL Anti-TGFB1 Beads.