Iridium DNA Intercalator (Fluidigm, 201192A) and metal-labeled antibodies pan-keratin (Nd 148, Fluidigm 3148020D), cytokeratin 7 (Dy 164, Fluidigm 3164028D), Ki-67 (Er 168, Fluidigm 3168022D), HLA-DR (Yb 174, Fluidigm 3174023D), α-SMA (Pr 141, Fluidigm 3141017D), vimentin (Nd 143, Fluidigm 3143029D), and collagen I (Tm 169, Fluidigm 3169023D) were purchased from Fluidigm. Tissue microarray (TMA) samples were purchased from BioMax (PA807). Recombinant mortalin was purchased from Novus Biologicals (NBC1-18380).
Iridium dna intercalator
Manufactured by Standard BioTools
Sourced in Australia
The Iridium DNA Intercalator is a lab equipment product that binds to DNA molecules. It functions by inserting itself between the base pairs of DNA, allowing for the detection and analysis of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Standard BioTools (1 mentions)
Nbc1 18380, by Novus Biologicals (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
W buffer, by Standard BioTools (1 mentions)
Cytof instrument, by Standard BioTools (1 mentions)
Anti cd45 30 f11, by BioLegend (1 mentions)
Anti cd11b m1 70, by BioLegend (1 mentions)
Anti ly6c hk1, by BioLegend (1 mentions)
Cytof, by Standard BioTools (1 mentions)
Eq four element calibration beads, by Standard BioTools (1 mentions)
Fc block, by BD (1 mentions)
Cisplatin, by Standard BioTools (1 mentions)
Helios mass cytometer, by Standard BioTools (1 mentions)
Protein transport inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Maxpar antibody labelling kit, by Standard BioTools (1 mentions)
6 protocols using iridium dna intercalator
1
Multiparametric Tissue Profiling with IMC
2
Comprehensive Single-Cell Cytometry Analysis
3
Immunohistochemical Analysis of Murine Brain
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Brains were isolated from mice perfused transcardially with PBS and 2% PFA. Brains were cut sagittally and placed in 2% PFA overnight, and subsequently placed sequentially in a series of sucrose solutions of increasing concentration (10%, 20%, and 30%). Murine brains were frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Tokyo, Japan) in hexane pre-chilled in liquid nitrogen. Frozen brain sections (8-9 μm) were fixed in methanol, rinsed in tris-buffered saline with 0.05% Tween 20 (TBST) and blocked with 10% FCS. Excess solution was shaken off before primary fluorophore-conjugated antibodies (anti-CD11b: M1/70, Biolegend and anti-NS1: 4G4, Roy Hall, UQ) were applied. Regions of interest were selected based on immunofluorescent staining visualised on the Olympus BX-51 microscope using a DP-70 camera and Cell Sensor software. Slides were then incubated with metal-conjugated antibodies overnight at 4 °C (anti-Ly6C (HK1.4, Biolegend), anti-CD86 (GL1, Becton Dickinson), anti-Iba1 (EPR165, Abcam), anti-CD45, (30-F11, Biolegend), anti-FITC (FIT-22, DVS) and anti-NeuN (Fox3, Biolegend)). Iridium DNA intercalator (Fluidigm, 1:2000 in TBST) was applied to brain sections, prior to sections being rinsed in UltraPure water and left to air dry. If slides were not immediately ablated on the Hyperion, they were stored at 4 °C until use.
4
High-Dimensional Immune Cell Profiling
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Cryo-preserved PBMCs were enriched for live cells by magnetic depletion of dead cells (Dead cells removal microbeads, Myltenyi) in presence of Citrate buffer. One Million cells per donor samples, healthy donor PBMCs and VeriCells were seeded in 96-well plate. Cells were washed, and each well was then stained with 100 μL of a unique double metal-labeled (Y89, Cd-106, Cd-110, Cd-112, Cd-116 and Cd-196) anti-CD45 antibody mix to further barcode the cells of individual donor. Cells were then washed twice, and five samples were combined into a single well. Cells were first stained with Fc block (BD Biosciences) during 10 min at room temperature. Cells were then washed and stained with the heavy metal-labeled antibody mixtures for 30 min on ice and 200 μM cisplatin during the last 5 min for the discrimination of live and dead cells. Cells were washed twice and fixed in 2% paraformaldehyde in PBS overnight at 4 °C. Cells were then washed and resuspended in 250 nM iridium DNA intercalator (Fluidigm) in 2% paraformaldehyde/PBS at room temperature. Cells were washed, pooled together, and adjusted to 0.5 million cells per milliliter in MaxPar water together with 10 % equilibration beads (EQ Four element calibration beads, Fluidigm) for acquisition at 250 events/ second on a HELIOS mass cytometer (CyTOF, Fluidigm).
5
Mass Cytometry Antibody Staining
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Antibodies were validated, pre-titered and supplied in pre-test amounts by the Ramaciotti Facility for Human Systems Biology Mass Cytometry Reagent Bank (University of Sydney, NSW, Australia). All steps until fixation were performed in the presence of Protein Transport Inhibitor Cocktail (Invitrogen, Thermo Fisher Scientific, NSW, Australia). Single cell suspensions were stained as previously described.46 Cells were incubated in 5 μM cisplatin (Fluidigm, NSW, Australia) for 5 min, then quenched with FACS wash and resuspended in surface stain monoclonal antibody mix listed in Supplementary Table 2 . Following staining, cells were fixed in 4% paraformaldehyde then permeabilised using the eBioscience™ Foxp3/ Transcription Factor Staining Buffer Set (eBiosciences, Thermo Fisher Scientific, NSW, Australia). Cells were then stained with antibodies, washed and stored overnight in 4% paraformaldehyde with 0.125 µM iridium DNA intercalator (Fluidigm, NSW, Australia). For analysis, cells were washed in FACS wash, resuspended to a final concentration of 106 cells/mL and samples acquired on a Helios Mass Cytometer (Fluidigm, NSW, Australia).
6
Maxpar Antibody Labeling for Tissue Staining
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A “Maxpar Antibody Labelling Kit” (Fluidigm) was used to conjugate a unique heavy metal element that is absent in the tissue to each of the antibodies (Table S1 ; protocol from Fluidigm, file PRD002 Version 11). Briefly, an antibody cocktail containing all eight conjugated antibodies each at the optimal concentration, as demonstrated in single‐plex immunofluorescence (Table S1 ), was incubated with tissue sections (12 hours, 22°C). Iridium DNA Intercalator (Fluidigm 201192) in 1:400 v/v TBST was incubated (5 minutes, 22°C) to stain nuclei within the myocardium.
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