Synovial Fibroblast 3.2: Secretion response of seven primary human synovial fibroblast samples from healthy and rheumatoid arthritis-diagnosed donors to a panel of 3 stimuli and 5 small molecule inhibitors (replicate 2 of 2) - Dataset (ID:20235)

HMS Dataset ID: 20235
Dataset Title: Synovial Fibroblast 3.2: Secretion response of seven primary human synovial fibroblast samples from healthy and rheumatoid arthritis-diagnosed donors to a panel of 3 stimuli and 5 small molecule inhibitors (replicate 2 of 2)
Publication(s) Using Dataset: PMID: 27820799
Project Summary Page(s): lincs.hms.harvard.edu/jones-natchembiol-2016
Screening Lab Investigator: Douglas S. Jones
Screening Principal Investigator: Peter Sorger, Douglas A. Lauffenburger
Assay Description: Supernatant levels of 51 cytokines, chemokines, growth factors, and proteases were measured using a multiplexed bead-based sandwich immunoassay to quantify the secretion response of 7 primary human synovial fibroblast samples from healthy and rheumatoid arthritis-diagnosed donors exposed to 3 different stimuli in the presence or absence of 1 of 5 small molecule kinase inhibitors. A full replicate of this experiment (including cell stimulation, supernatant recovery, and Luminex analysis) was conducted on a separate day and is available in HMS dataset #20234. A third, related dataset from the same project also is available: #20233.
Assay Protocol: 1. Primary human synovial fibroblasts (SF) from 3 normal donors (human fibroblast-like synovioctyes (HFLS), cat. no. 408-05a) and 4 rheumatoid arthritis (RA) donors (HFLS-RA, cat. no. 408RA-05a) were purchased from Cell Applications, Inc. The cells were cultured according to the supplier’s recommendations using Synoviocyte Growth Medium (Cell Applications, Inc., cat. no. 415-500) as a full growth medium. Cells were provided at passage 2, and all experiments were conducted on cells at passage 3-6 in accordance with published recommendations (Rosengren et al, 2007).
2. The cells were seeded (1000 cells/well) into 384-well plates (Costar, cat. no. 3712).
3. Following overnight incubation in full growth medium at 37°C and 5% CO2, the cells were starved in basal medium (Synoviocyte Basal Medium; Cell Applications, Inc., cat. no. 414-500) overnight (~16 hr) followed by an additional starving step in basal medium approximately 4 hr prior to exposure to stimulatory factors.
4. For inhibitor treatments, the cells were pretreated with an inhibitor or with DMSO as a control for 3 hr prior to stimulation. Inhibitors were used at their ~IC95 concentration as determined by inhibition of a proximal downstream signaling target. This corresponded to 3 μM JNK-IN-8 (determined by inhibition of TNFα-induced p-c-Jun), 0.6 μM PH-797804 (determined by inhibition of TNFα-induced p-Hsp27), 2 μM IKK16 (determined by inhibition of TNFα-induced NFκB nuclear translocation), 0.3 μM tofacitinib (determined by inhibition of IL-6-induced p-STAT1 and p-STAT3), and 0.6 μM 5z-7-oxozeaenol (determined by inhibition of IL-1α- or TNFα-induced inhibition of NFκB translocation, p-c-Jun, and p-Hsp27).
5. Following 18 hr stimulation, supernatants were recovered and clarified by 10 min centrifugation at 2000 RPM. Supernatants from two adjacent wells (e.g. wells A1 and A2, which comprised biological replicates) were pooled. Downstream statistical analyses considered data from pooled supernatants as single replicates. Pooled supernatants were adjusted with 1x phosphate buffered saline (PBS) and 5% bovine serum albumin (BSA) to contain 0.25% final concentration BSA, clarified again by centrifugation, aliquoted, and stored at -80°C. The full experiment was repeated on a separate day and is presented in dataset #20234.
6. Multiplexed bead-based immunoassays used reagents purchased from Bio-Rad and R&D Systems and were analyzed on a Flexmap 3D using xPONENT software (Luminex Corp.). The levels of secreted cytokines were measured using two sets of Bio-Rad detection panels: group I 27-plex (Bio-Rad, cat. no. M500KCAF0Y) and group II 21-plex (Bio-Rad, cat. no. MF0005KMII) human cytokine panels. Levels of MMP-1, -2, and -3 were measured using the Luminex Peformance Human MMP Panel (R&D Systems, cat. nos. LMP901B, LMP901C, and LMP513B). Supernatants either were diluted 1:3 with 1xPBS / 0.05% BSA / 0.05% Tween-20 (for the Bio-Rad cytokine kits) or diluted 1:5 with Calibrator Diluent RD5-37 buffer (for the R&D Systems MMP Panel; this buffer is from the Human MMP Base Kit, cat. no. LMP000B) and assayed according to the supplier’s instructions alongside a 10-point standard serial dilution to establish the dynamic range of the assay for each analyte. The immunoassay results are reported as the median fluorescent intensity (MFI) across all beads measured per sample. The number of beads per well (bead count) also was measured to account for sampling error. Lastly, the level of cross-reactivity between stimuli and analyte was quantified and accounted for by individually spiking control samples containing ligands at the same concentration as the stimulating condition on the same Luminex plate as the cell-stimulated supernatants.
Assay Protocol Reference: Rosengren, S., Boyle, D. L. & Firestein, G. S. (2007) Acquisition, culture, and phenotyping of synovial fibroblasts. Methods Mol. Med. 135, 365–375.
HMS Dataset Type: Bead-based immunoassay
Date Publicly Available: 2015-08-27
Most Recent Update: 2016-11-08