Cells collected from secondary lymph organs or CSF of patients were stained with fluorescent-conjugated antibody: fixable viability dye (Thermo Fisher Scientific, Cat# 65-0866-14), CD45 (BioLegend, Cat# 103132), CD3 (BioLegend, Cat# 100213), CD8 (Thermo Fisher Scientific, Cat# 53-0081-82, 17-0088-42), CD4 (Thermo Fisher Scientific, Cat# 62-00420-80), CD11b (BioLegend, Cat# 101222), Ly6G (BioLegend, Cat# 127613), Ly6C (BioLegend, Cat# 128017), CD11c (BioLegend, Cat# 117322), H-2Kb bound to SIINFEKL (BioLegend, Cat# 141605), OVA pentamer (ProImmune, Cat# F93-2A-G), SA-β-gal (Dojindo, Cat# SG03), and CD49d (Thermo Fisher Scientific, Cat# 12-0492-81, 16-0492-85) for 30 min at 4℃; primary antibodies: luciferase (Abcam, Cat# ab185924, 1:100), cleaved caspase-3 (Cell Signaling Technology, Cat# 9661, 1:200), and Ki-67 (Thermo Fisher Scientific, Cat# 14-5698-80, 1:50). For intracellular staining, cells were pretreated with Foxp3/Transcription Factor Fixation/Permeabilization kit (eBioscience, Cat# 00-5521-00) according to the manufacturer’s instructions. Flow cytometry was performed on Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software.
H 2kb bound to siinfekl
Manufactured by BioLegend
H-2Kb bound to SIINFEKL is a lab equipment product that serves as a major histocompatibility complex (MHC) class I molecule. It presents the SIINFEKL peptide, which is derived from the chicken ovalbumin protein. This product can be used for various research applications where the function of MHC class I molecules and their interactions with peptides need to be studied.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cellrox reagent, by Thermo Fisher Scientific (2 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Sa β gal, by Dojindo Laboratories (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cd11b, by BioLegend (1 mentions)
Lsr fortessa instrument, by BD (1 mentions)
2 protocols using h 2kb bound to siinfekl
1
Multi-Parametric Immune Cell Profiling
2
Cell Surface Protein and ROS Assay
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To measure the expression of MHC-I and PD-L1 on the cell surface, we plated cells in a 6-well format in triplicates as described above. Cells were harvested with trypsin, rinsed, and 1 million cells were resuspended in PBS containing 1% fetal bovine serum (ThermoFisher Scientific). Fluorescently-labeled antibodies against MHC-I (Cat# 311406 and 114608), H-2KB bound to SIINFEKL (Cat# 141604), and PD-L1 (Cat# 393610 and 124312) (Biolegend, San Diego, CA) were added at a final dilution of 1:100. Cells were counter-stained with DAPI at a final concentration of 1 ng/mL and analyzed on a BD LSR Fortessa instrument (BD Biosciences, Franklin Lakes, NJ). The resulting data was processed using the software FlowJo (BD Biosciences). For the assessment of reactive oxygen species, cells were treated with CTL and MR media as above. After 24h, plated cells were incubated with CellROX reagent (ThermoFisher) at a final concentration of 2 μM in PBS for 30 min at 37°C. Cells were then harvested and analyzed as above.
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