Monocytes were purified by negative selection with a Classical Monocyte Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. Purity exceeded 80%. DLBCL cells were incubated in bottom compartments of 8-µM 24-well Transwell plates (Nunc) at 0.5 × 106 cells per milliliter. Twenty-four hours later, 0.2 × 106 purified monocytes were added to upper compartments. After 2 hours of incubation, cells from the bottom compartment were stained for CD14, and CD14+ monocytes were enumerated by flow cytometry with an Accuri C6 (BD Biosciences) set on a volume-based event acquisition. Flow variations were controlled by the addition of fluorescent beads (BD Biosciences). The invasion assay was performed similarly but the filter was covered with 50 µL of Matrigel (BD Biosciences), and the incubation time was extended to 18 hours. Polyclonal goat anti-CCL5 (R&D Systems) and control goat IgG (Sigma) were used at 10 µg/mL. metCCL5 was used at 100 ng/mL. Maraviroc and BX471 (Sigma) were used at 100 µM and 100 nM, respectively.
Control goat igg
Manufactured by Merck Group
Control goat IgG is a laboratory reagent used as a control in immunoassays and other applications involving goat immunoglobulins. It provides a consistent reference point for evaluating experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent beads, by BD (1 mentions)
Maraviroc, by Merck Group (1 mentions)
Classical monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Bx471, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Control mouse igg, by Merck Group (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
2 protocols using control goat igg
1
Monocyte Invasion Assay with DLBCL
2
Depletion of IGFBP Proteins from MSC-CM
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To deplete IGFBP‐3, IGFBP‐4, and IGFBP‐6 from MSC‐CM, immunoprecipitation was performed. Briefly, MSC‐CM was incubated with antibody conjugated to protein G‐Sepharose (GE Healthcare) for 24 h at 4°C. The following primary antibodies were used: goat anti‐human IGFBP‐3 (#AF675; R&D Systems), mouse anti‐human IGFBP‐4 (clone #82314R; R&D Systems), mouse anti‐human IGFBP‐6 (clone #164428; R&D Systems), control goat IgG (Sigma‐Aldrich), and control mouse IgG (Sigma‐Aldrich). After centrifugation, the supernatant was filtered with a 0.22 μm filter and used for subsequent experiments. The extent of depletion of IGFBP‐3, IGFBP‐4, and IGFBP‐6 were confirmed by measuring the residual concentration by respective enzyme‐linked immunoassays (ELISAs) and determined to be approximately 80%, 60%, and 50%, respectively.
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