Milliplex map kit magnetic bead panels

Luminex cytokine assays was performed following the manufacturer’s directions on serum samples isolated prior to infection and at the terminal bleed [Fig 1]. Briefly, undiluted serum samples isolated from humanized mice infected with HTLV-1, S. stercoralis, or both HTLV-1 and S. stercoralis were analyzed using Milliplex Map Kit magnetic bead panels as per the manufacturer’s protocol (EMDMillipore). Plates were analyzed on a MAGPIX Luminex machine (Austin, TX, USA). All analyte concentrations were calculated using Milliplex Analyst software (EMDMillipore). The following 8 human cytokines were measured in the serum of the control and infected animals: IL-4, IL-5, IL-10, IL-12p40, IL-13, IL-17A, IFN-γ and TNF-α.

Fluid recovered from the diffusion chambers from each mouse was analyzed using Milliplex map Kit magnetic bead panels per manufacturer’s protocols (EMDMillipore) and a MAG-PIX Luminex multiplexing instrument (Luminex, Austin, TX). Diffusion chamber fluid was analyzed for 11 cytokines (IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, GM-CSF and G-CSF) and 9 chemokines (eotaxin, RANTES, KC, MCP-1, IP-10, MIG/CXCL9, MIP-1α, MIP-1β and MIP-2). Analyte concentrations were calculated using Milliplex Analyst software (EMDMillipore).

Spleens were collected aseptically at the termination of the experiment, kept on ice in 0.1% BSA and homogenized into a single cell suspension. Following lysis of erythrocytes using sterile distilled cold water, the suspension was filtered through a 70 μm cell strainer and washed using Dulbecco’s Modified Eagle medium (Corning). Cells were cultured in flat-bottom 96-well plates, with each well containing 2×10⁶ cells. The cells were stimulated with 10 μg of either Ov-103 or Ov-RAL-2 for 72 hours at 37 °C. IL-4 uptake by splenocytes was blocked by addition of 0.5 μl of anti-IL-4R (BD Biosciences) to each well. Supernatants were collected following incubation and frozen at −20 °C. Cytokines in the supernatants were analyzed using Milliplex map Kit magnetic bead panels per manufacturer’s protocols (EMDMillipore) and a MAG-PIX Luminex multiplexing instrument (Luminex, Austin, TX). Stimulated spleen cell supernatants were analyzed for 14 different cytokines: IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-21, IL-23, IL-27, IL-33, IL-17A, IL-17E and IL-17F. Analyte concentrations were calculated using Milliplex Analyst software (EMDMillipore).