Iridium intercalator solution

Manufactured by Standard BioTools

The Iridium intercalator solution is a laboratory reagent designed for use in various biological and analytical applications. It functions as an intercalating agent, allowing it to bind to DNA or RNA molecules. This solution can be utilized in techniques such as flow cytometry and microscopy to stain and visualize nucleic acid samples.

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Lab products found in correlation

Permeabilization buffer, by Thermo Fisher Scientific (7 mentions) Sodium azide, by Merck Group (6 mentions) Bovine serum albumin (bsa), by Merck Group (6 mentions) Fix perm buffer, by Thermo Fisher Scientific (6 mentions) Ddh2o, by Standard BioTools (6 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (5 mentions) Saponin, by Merck Group (4 mentions) Phosphate buffered saline (pbs), by Merck Group (3 mentions) Cisplatin, by Merck Group (3 mentions) Permeabilization buffer, by Merck Group (2 mentions) Maxpar water, by Standard BioTools (2 mentions) Foxp3 fixation permeabilization buffer, by BioLegend (2 mentions) Helios cytof mass cytometer, by Standard BioTools (2 mentions) Foxp3 fix perm buffer set, by BioLegend (1 mentions) Five element beads, by Standard BioTools (1 mentions) Maxpar x8 kit, by Standard BioTools (1 mentions) Methanol free formaldehyde solution, by Thermo Fisher Scientific (1 mentions) 70 m cell strainer, by BD (1 mentions) Cell id cisplatin, by Standard BioTools (1 mentions) Maxpar reagent kits, by Standard BioTools (1 mentions)

18 protocols using iridium intercalator solution

Intracellular Antibody Staining for Mass Cytometry and Flow Cytometry

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For mass cytometry, the combined surface- and live/dead-stained sample was fixed with 1.6% paraformaldehyde in PBS (PFA; Electron Microscopy Sciences) for 30 min at 4 °C on a rotary shaker (500 rpm). Fixed cells were washed twice with permeabilization buffer (PBS, 0.5% saponin, 2% BSA and 0.01% sodium azide, (all Sigma)). Cells were resuspended in 400 μL of intracellular antibody mixture (Table S2) in permeabilization buffer for 1 h at 4 °C on a rotary shaker (500 rpm). The sample was washed, supernatant removed and the cells resuspended in 1x iridium intercalator solution (Fluidigm) over night. Lastly, the sample was washed twice with PBS/BSA, once with ddH2O before acquisition. For flow cytometry, similar fixation and permeabilization was performed. Cells were resuspended in 50 μL of intracellular antibody mixture (Table S8) in permeabilization buffer for 1 h at 4 °C on a rotatory shaker. Samples were then washed with CSM.

Galli E., Hartmann F.J., Schreiner B., Ingelfinger F., Arvaniti E., Diebold M., Mrdjen D., van der Meer F., Krieg C., Al Nimer F., Sanderson N., Stadelmann C., Khademi M., Piehl F., Claassen M., Derfuss T., Olsson T, & Becher B. (2019). GM-CSF and CXCR4 Define a T Helper Cell Signature in Multiple Sclerosis. Nature medicine, 25(8), 1290-1300.

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Comprehensive Antibody Staining for Mass Cytometry

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Antibodies used for these studies can be found in Supplemental Table 4. Antibodies for mass cytometry or flow-cytometry were purchased pre-conjugated to a heavy metal (Fluidigm), biotin, or a fluorochrome (Biolegend). To identify dead cells, lung samples were previously incubated with 200 nM cisplatin (Sigma-Aldrich) for 5 minutes at room temperature (RT). Surface molecules were then stained with primary antibodies for 30 minutes at RT, followed by incubation with metal-conjugated secondary antibodies when needed. After surface staining, cells were fixed with 1.6% paraformaldehyde (PFA) in PBS for 20 minutes at RT, and then fixed and permeabilized with the Foxp3 fix/perm buffer set (Biolegend) for 45 minutes at RT. Cells were washed 2 times with perm wash, and incubated with anti-FOXP3, anti-CD68, anti-CD56, anti-CD4 and anti-CD8 (the surface markers used here were undetectable after tissue digestion, and an intracellular staining was used as an alternative method), for 30 minutes at RT. Cells were washed and resuspended in 1x iridium intercalator solution (Fluidigm) overnight. Next, samples were washed in PBS and then in distilled water and acquired using Helios mass cytometry at Vanderbilt University Mass Cytometry Center of Excellence. Acquisition was performed in the same day and all samples were normalized using five-element beads (Fluidigm).

Richmond B.W., Mansouri S., Serezani A., Novitskiy S., Blackburn J.B., Du R.H., Fuseini H., Gutor S., Han W., Schaff J., Vasiukov G., Xin M.K., Newcomb D.C., Jin L., Blackwell T.S, & Polosukhin V.V. (2020). Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD. Mucosal Immunology, 14(2), 431-442.

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Single-Cell Intracellular Antibody Staining

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The cells were washed twice with permeabilization buffer [0.5% saponin, 2% bovine serum albumin, and 0.01% sodium azide (all Sigma-Aldrich) in PBS]. Cells were resuspended in 400 μL intracellular antibody mixture in permeabilization buffer for 1 h at 4 °C on a rotary shaker (500 rpm). The samples were then washed, the supernatant removed, and the cells resuspended in 1X iridium intercalator solution (Fluidigm) overnight. Finally, the sample was washed twice with PBS/bovine serum albumin and once with double-distilled water before acquisition.

Liu X., Chen B., Huang Z., Duan R., Li H., Xie L., Wang R., Li Z., Gao Y., Zheng Y, & Su W. (2021). Effects of poor sleep on the immune cell landscape as assessed by single-cell analysis. Communications Biology, 4, 1325.

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Multiparametric Immune Cell Profiling

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The barcoded sample convolute was labeled in 400 μl CSM containing the antibody mix directed against surface markers for 40 min at 37 °C on an orbital shaker (500 rpm). For dead cell discrimination, 2.5 μM cisplatin (Sigma-Aldrich) was added for 2 min on ice.
For transcription factor detection, the sample convolute was fixed and permeabilized for 40 min at 4 °C in 1X FOXP3 Fixation/Permeabilization Buffer (BioLegend). Sample was washed in permeabilization buffer [(PBS, 0.5% saponin, 2% bovine serum albumin (BSA), 0.01% sodium azide (all Sigma-Aldrich)] and nuclear staining was performed in 400 μl permeabilization buffer for 1 h at 4 °C.
For intracellular cytokine detection the sample convolute was fixed in 1.6% paraformaldehyde (Electron Microscopy Sciences) for 1 h at 4 °C. The convolute was washed with permeabilization buffer and labeled with antibodies recognizing intracellular cytokines in 400 μl permeabilization buffer for 1 h at 4 °C.
In both the cases, the labeled and stained sample mix was washed and resuspended in 1X iridium intercalator solution (Fluidigm) followed by a 4 °C incubation overnight. Finally, the sample was washed twice with PBS and twice with Max Par water (Fluidigm) before data acquisition.

Ingelfinger F., Krishnarajah S., Kramer M., Utz S.G., Galli E., Lutz M., Zwicky P., Akarca A.U., Jurado N.P., Ulutekin C., Bamert D., Widmer C.C., Piccoli L., Sallusto F., Núñez N.G., Marafioti T., Schneiter D., Opitz I., Lanzavecchia A., Jung H.H., De Feo D., Mundt S., Schreiner B, & Becher B. (2021). Single-cell profiling of myasthenia gravis identifies a pathogenic T cell signature. Acta Neuropathologica, 141(6), 901-915.

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Intracellular Antibody Staining for Mass Cytometry and Flow Cytometry

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CyTOF-based Immunophenotyping with Barcoding

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After cell barcoding, washing and pelleting, the combined samples were stained and processed as described previously^{17 (link)}. Briefly, cells were re-suspended in 100 µl of antibody cocktail directed against cell surface markers (Supplementary Tables 2 and 3) and incubated for 30 minutes at 4 °C. Then, cells were washed twice with cell staining buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA). For intracellular staining, the stained (non-stimulated) cells were then incubated in fixation/permeabilization buffer (Fix/Perm Buffer, eBioscience) for 60 minutes at 4 °C. Cells were then wash twice with permeabilization buffer (eBioscience). The samples were then stained with antibody cocktails directed against intracellular molecules (Supplementary Tables 2 and 3) in permeabilization buffer for 1 hour at 4 °C. Cells were subsequently washed twice with permeabilization buffer and incubated overnight in 4% methanol-free formaldehyde solution. The fixed cells were then washed and re-suspended in 1 ml iridium intercalator solution (Fluidigm) for 1 hour at room temperature, followed by two washes with cell staining buffer and two washes with ddH₂O (Fluidigm). Finally, cells were pelleted and kept at 4 °C until CyTOF measurement.

Böttcher C., Fernández-Zapata C., Schlickeiser S., Kunkel D., Schulz A.R., Mei H.E., Weidinger C., Gieß R.M., Asseyer S., Siegmund B., Paul F., Ruprecht K, & Priller J. (2019). Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis. Scientific Reports, 9, 19471.

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Single-Cell Multiparametric Cytometry Protocol

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After cell barcoding, washing and pelleting, the combined samples were stained and processed as described previously [16 (link), 17 (link)]. Briefly, cells were re-suspended in 100 μl of antibody cocktail directed against cell surface markers (Additional file 1: Supplementary Tables 7 and 8) and incubated for 30 min at 4 °C. Then, cells were washed twice with cell staining buffer (PBS containing 0.5% BSA and 2 mM EDTA). For intracellular staining, the stained (non-stimulated) cells were then incubated in fixation/permeabilization buffer (Fix/Perm Buffer, eBioscience) for 60 min at 4 °C. Cells were then wash twice with permeabilization buffer (eBioscience). The samples were then stained with antibody cocktails directed against intracellular molecules (Additional file 1: Supplementary Tables 7 and 8) in permeabilization buffer for 1 h at 4 °C. Cells were subsequently washed twice with permeabilization buffer and incubated overnight in 4% methanol-free formaldehyde solution. The fixed cells were then washed and re-suspended in 1 ml iridium intercalator solution (Fluidigm) for 1 h at RT, followed by two washes with cell staining buffer and two washes with ddH₂O (Fluidigm). Finally, cells were pelleted and kept at 4 °C until CyTOF measurement.

Böttcher C., van der Poel M., Fernández-Zapata C., Schlickeiser S., Leman J.K., Hsiao C.C., Mizee M.R., A.d.e.l.i.a., Vincenten M.C., Kunkel D., Huitinga I., Hamann J, & Priller J. (2020). Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis. Acta Neuropathologica Communications, 8, 136.

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Intracellular Antibody Staining Protocol

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The cells were washed twice with permeabilization buffer (0.5% saponin, 2% bovine serum albumin, and 0.01% sodium azide [all MilliporeSigma] in PBS). Then we resuspended the cells in 50 μL of intracellular antibody mixture in permeabilization buffer for 1 hour on a rotary shaker at 4°C. Then we washed the samples, removed the supernatant, and resuspended the cells in 1× iridium intercalator solution (Fluidigm) overnight. At last, the sample was washed twice with PBS/bovine serum albumin and once with double-distilled water before acquisition.

Liu X., Jiang Q., Lv J., Yang S., Huang Z., Duan R., Tao T., Li Z., Ju R., Zheng Y, & Su W. (2022). Insights gained from single-cell analysis of immune cells in tofacitinib treatment of Vogt-Koyanagi-Harada disease. JCI Insight, 7(23), e162335.

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Barcoding Enhances Single-Cell Proteomics

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A live‐cell barcoding method was used to decrease inter‐sample staining variability. The barcoded and combined samples were stained with 0.5 μmol/L cisplatin‐195pt. The cells were re‐suspended in pre‐cooled Maxpar Cell Staining Buffer and washed with PBS/bovine serum albumin. For surface staining, the cells were added to a surface antibody mixture and incubated at 37°C for 30 min. The samples were stored in 2% formaldehyde in PBS at 4°C overnight. Then, the cells were washed and re‐suspended in intracellular antibodies mixture for 1 h at 4°C. The samples were then added to iridium intercalator solution (Fluidigm) overnight.

Liu X., Su Y., Huang Z., Lv J., Gu C., Li Z., Tao T., Liu Y., Jiang Q., Duan R., Chen B., Ju R., Wang X., Zheng Y, & Su W. (2023). Sleep loss potentiates Th17‐cell pathogenicity and promotes autoimmune uveitis. Clinical and Translational Medicine, 13(5), e1250.

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Multiparametric Analysis of Immune Cells

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Anti-human antibodies (Supplementary Table 9) were purchased either preconjugated to metal isotopes (Fluidigm) or from commercial suppliers in purified form and conjugated in-house using the MaxPar X8 kit (Fluidigm) according to the manufacturer’s protocol. For surface and intracellular staining, after cell barcoding, washing and pelleting, the combined samples were resuspended in 100 µl antibody cocktail against surface markers (Supplementary Table 9) and incubated for 30 min at 4 °C. Then, cells were washed twice with cell staining buffer. For intracellular staining, the stained (non-stimulated) cells were then incubated in fixation/permeabilization buffer (Fix/Perm Buffer, eBioscience) for 60 min at 4 °C. Cells were then washed twice with permeabilization buffer (eBioscience). The samples were then stained with antibody cocktails against intracellular molecules in permeabilization buffer for 1 h at 4 °C. Cells were subsequently washed twice with permeabilization buffer and incubated overnight in 2% methanol-free formaldehyde solution. Fixed cells were then washed and resuspended in 1 ml iridium intercalator solution (Fluidigm) for 1 h at RT. Next, the samples were washed twice with cell staining buffer and then twice with ddH₂O (Fluidigm). Cells were pelleted and kept at 4 °C until CyTOF measurement.

Sankowski R., Süß P., Benkendorff A., Böttcher C., Fernandez-Zapata C., Chhatbar C., Cahueau J., Monaco G., Gasull A.D., Khavaran A., Grauvogel J., Scheiwe C., Shah M.J., Heiland D.H., Schnell O., Markfeld-Erol F., Kunze M., Zeiser R., Priller J, & Prinz M. (2023). Multiomic spatial landscape of innate immune cells at human central nervous system borders. Nature Medicine, 30(1), 186-198.

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