human igg1 fc control  (R&D Systems)

Journal: bioRxiv

Article Title: NKG2D receptor ligands are cell surface biomarkers for injured murine and human nociceptive sensory neurons

doi: 10.1101/2025.07.16.665122

Figure Lengend Snippet: A) Bright-field image of uninjured iPSC-derived sensory neurons. B) βtubIII ( magenta ) and C) NKG2D-Fc ( green ) immunolabelling of iPSC sensory neurons (‘840’ line) without injury. D) Quantification of NKG2D-Fc receptor particle density on βtubIII+ axons compared to Fc-only controls. E) Bright-field image of iPSC-derived sensory neurons after axonal injury by laser ablation. F ) βtubIII ( magenta ) and G ) NKG2D-Fc ( green ) immunolabelling of iPSC sensory neurons (‘840’ line) 7 days after laser ablation. Arrows highlight axons labelled with NKG2D. H ) Quantification of NKG2D-Fc receptor particle density on βtubIII+ axons compared to Fc-only controls. n=3 experimental repeats, 2 wells per group, per repeat. Two-way ANOVA, NKG2D versus Fc: F (1, 8) = 8.542, *p=0.0192. Sidak’s multiple comparison test: *p=0.0442. I ) Photo of microfluidic hiPSCd sensory neuron cultures. Sensory neuron precursors (i.e. neuronal soma) seeded in left hand chamber. Axons grow through microfluidic channels into neurite chamber on right hand side. Inset ) High magnification of microfluidic channels. J ) βtubIII immunolabelling of iPSC sensory axons in neurite compartment. Eight regions of interest ( white squares ) were sampled for proximal and distal axon/neurite quantification. K ) Example ROI of distal axons/neurites from microfluidic devices treated with freshy thawed (control, left ) or IL-2 primed human NK cells ( right ). NK cells in gre en. L ) Quantification of axon/neurite fragmentation. n=3 experimental repeats (6 microfluidic devices; n=8 ROI per device) with NK cells from three different healthy donors; sensory neurons derived from iPSC donor ‘AD2’. Proximal axon fragmentation ns, p=0.1610 (t=1.717); Distal axon fragmentation **p=0.0079 (t=4.931), unpaired t test. M ) Example ROI of distal axons/neurites from microfluidic devices treated with freshy thawed (unstimulated), IL-2 stimulated NK cells incubated with IgG1, IL-2 stimulated NK cells incubated with anti-NKG2D or not treated with NK cells. NK cells in blue (DAPI), axons in magenta (BtubIII). N ) Microfluidics with different number of NK cells (50×10 3 , 100×10 3 , 200×10 3 ) were included for quantification of axon/neurite fragmentation. n=3 experimental repeats (3 microfluidic devices; n=10 ROI per device) with NK cells from three different healthy donors; sensory neurons derived from iPSC donor ‘840’. Multiple comparisons were analysed by two-way ANOVA with Šídák correction. Adjusted p=0.0106 for comparison between microfluidics with unstimulated NK cells and IL-2-stimulated NK cells with IgG1, NK=100×10 3 ; adjusted p=0.0009 for comparison between microfluidics with unstimulated NK cells and IL-2-stimulated NK cells with IgG1, NK=200×10 3 ; **adjusted p=0.0036 for comparison between microfluidics with IL2-stimulated NK cells incubated with IgG1 and anti-NKG2D, NK=200×10 3 . Scale bars as indicated.

Article Snippet: Recombinant murine NKG2D-Fc chimeric receptor (Cat. 139-NK, R&D Systems; lot numbers: FRP0218081, FRP0222021, FRP0223061, FRP0224041), recombinant human NKG2D-Fc chimeric receptor (Cat: 1299-NK, R&D Systems; lot number: FVV0618021) and recombinant human IgG1 Fc control (Cat. 110-HG, R&D Systems, lot number: EAX0619041) were prepared by suspension in distilled water at 1 mg/ml for 15 min at room temperature (RT) and further diluted to 100 μg/ml in phosphate buffered saline (PBS) containing 0.1% BSA for storage at −80°C.

Techniques: Derivative Assay, Comparison, Control, Incubation