Iridium containing intercalator

Manufactured by Standard BioTools

The Iridium-containing intercalator is a laboratory reagent used in various analytical techniques. It functions as a fluorescent stain that binds to nucleic acids, allowing for the detection and visualization of DNA and RNA in biological samples.

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Lab products found in correlation

Normalization beads, by Standard BioTools (1 mentions) Helios mass cytometer, by Standard BioTools (1 mentions)

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Iridium-containing DNA intercalator Iridium intercalator Iridium

7 protocols using iridium containing intercalator

Single-Cell Barcoding and Immunophenotyping

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Cell samples in methanol were washed three times with Cell Staining Media (CSM, PBS with 0.5% BSA, 0.02% NaN₃) and once with PBS at 4°C. The cells were then re-suspended at 1 million cells/ml in PBS containing barcoding reagents (¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd, ¹¹⁰Pd, ¹¹³In and ¹¹⁵In,) each at a final concentration of 100 nM. Cells and barcoding reagent were incubated for 30 minutes at room temperature. Barcoded cells were then washed three times with CSM, pooled and stained with the metal-conjugated antibody mix (S1 Table) at room temperature for 1 hour. Unbound antibodies were removed by washing cells three times with CSM and once with PBS. For cellular DNA staining, an iridium-containing intercalator (Fluidigm) was diluted to 250 nM in PBS containing 1.6% PFA and added to the cells at 4°C for overnight incubation. Before measurement, the intercalator solution was removed and cells were washed with CSM, PBS, and ddH₂O. After the last washing step, cells were resuspended in MilliQ H₂O to 1 million cells/ml and filtered through a 40-μm strainer.

Krishnaswamy S., Zivanovic N., Sharma R., Pe’er D, & Bodenmiller B. (2018). Learning time-varying information flow from single-cell epithelial to mesenchymal transition data. PLoS ONE, 13(10), e0203389.

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Multiplexed Single-Cell Barcoding

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Formalin-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incubated in PBS containing barcoding reagents of ⁸⁹Y (100 nM), ¹⁰³Rh (2 μM), ¹⁰⁵Pd (100 nM), ¹⁰⁶Pd (100 nM), ¹⁰⁸Pd (100 nM), ¹¹⁰Pd (100 nM), ¹¹³In (200 nM), ¹¹⁵In (100 nM), and ²⁰⁹Bi (20 nM) for 30 min at room temperature and then washed three times with CSM. Barcoded cells were then pooled and stained with the metal-conjugated antibody mix (Table S2) at room temperature for 1 h. The antibody mix was removed by washing cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) diluted in PBS with 1.6% PFA was incubated with the cells at 4°C overnight. On the day of the measurement, the intercalator solution was removed, and cells were washed with CSM, PBS, and ddH₂O. After the last washing step, cells were resuspended in ddH₂O and filtered through a 70-μm strainer.

Lun X.K., Szklarczyk D., Gábor A., Dobberstein N., Zanotelli V.R., Saez-Rodriguez J., von Mering C, & Bodenmiller B. (2019). Analysis of the Human Kinome and Phosphatome by Mass Cytometry Reveals Overexpression-Induced Effects on Cancer-Related Signaling. Molecular Cell, 74(5), 1086-1102.e5.

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Single-Cell Barcoding and Staining

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Cell samples in methanol were washed three times with CSM (PBS with 0.5% BSA, 0.02% NaN₃) and once with PBS at 4 °C. The cells were then resuspended at 1 × 10⁶ cells ml⁻¹ in PBS containing barcoding reagents (¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd and ¹¹⁰Pd; Fluidigm) were conjugated to bromoacetamidobenzyl-EDTA (BABE, Dojindo) and two indium isotopes (¹¹³In and ¹¹⁵In, Fluidigm) were conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid 10-maleimide ethylacetamide (mDOTA, Mycrocyclics) following standard procedures^{51 (link),52}. Cells and barcoding reagent were incubated for 30 min at room temperature. Barcoded cells were then washed three times with CSM, pooled and stained with the metal-conjugated antibody mix (Supplementary Table 2) at room temperature for 1 h. Unbound antibodies were removed by washing cells three times with CSM and once with PBS. For cellular DNA staining, an iridium-containing intercalator (Fluidigm) was diluted to 250 nM in PBS containing 1.6% PFA, added to the cells at 4 °C, and incubated overnight. Before measurement, the intercalator solution was removed and cells were washed with CSM, PBS and doubly distilled H₂O. After the last wash step, cells were resuspended in MilliQ H₂O to 1 × 10⁶ cells ml⁻¹ and filtered through a 40-μm strainer.

Chen W.S., Zivanovic N., van Dijk D., Wolf G., Bodenmiller B, & Krishnaswamy S. (2020). Uncovering axes of variation among single-cell cancer specimens. Nature methods, 17(3), 302-310.

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CyTOF Cell Barcoding and Staining

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Formalin-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incubated in PBS containing barcoding reagents (¹⁰²Pd,¹⁰⁴Pd,¹⁰⁵Pd,¹⁰⁶Pd,¹⁰⁸Pd,¹¹⁰Pd,¹¹³In, and¹¹⁵In) at a final concentration of 100 nM for 30 min at room temperature and then washed three times with CSM (Bodenmiller et al., 2012 (link)). Barcoded cells were then pooled and stained with the metal-conjugated antibody mix (Supplementary Table 4) at room temperature for 1 h. The antibody mix was removed by washing cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) diluted in PBS with 1.6% PFA was incubated with the cells at 4°C overnight. On the day of the measurement, the intercalator solution was removed, and cells were washed with CSM, PBS, and doubly distilled H₂O. After the last washing step, cells were resuspended in doubly distilled H₂O and filtered through a 70-μm strainer.

Wade J.D., Lun X.K., Zivanovic N., Voit E.O, & Bodenmiller B. (2020). Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition. Frontiers in Physiology, 11, 579117.

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High-dimensional Immune Profiling by Mass Cytometry

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Post-thaw, fixed samples were added to an erythrocyte lysis buffer (SmartTube) and underwent two rounds of erythrocyte lysis. Cells were then barcoded as previously described^{84 (link)}. In summary, 20-well barcode plates were prepared with a combination of two Pd isotopes out of a pool of six (¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd, ¹¹⁰Pd) and added to the cells in 0.02% saponin/phosphate buffered saline. Samples were pooled and stained with metal-conjugated antibodies collectively to minimize experimental variation. The panels for the different cohorts are listed in Supplementary Tables 3 and 4. Intracellular staining was performed in methanol-permeabilized cells. Cells were incubated overnight at 4 °C with an iridium-containing intercalator (Fluidigm). Before mass cytometry analysis, cells were filtered through a 35-μm membrane and resuspended in a solution of normalization beads (Fluidigm).
Barcoded and stained cells were analysed on a Helios mass cytometer (Fluidigm) at an event rate of 500 to 1,000 cells per second. The data were normalized using Normalizer v0.1 MATLAB Compiler Runtime^{85 (link)} and debarcoded with a single-cell MATLAB debarcoding tool^{84 (link)}. Gating was performed using Cytobank (cytobank.org). Gating strategies for the different cohorts are shown in Supplementary Fig. 4.

Culos A., Tsai A.S., Stanley N., Becker M., Ghaemi M.S., McIlwain D.R., Fallahzadeh R., Tanada A., Nassar H., Espinosa C., Xenochristou M., Ganio E., Peterson L., Han X., Stelzer I.A., Ando K., Gaudilliere D., Phongpreecha T., Marić I., Chang A.L., Shaw G.M., Stevenson D.K., Bendall S., Davis K.L., Fantl W., Nolan G.P., Hastie T., Tibshirani R., Angst M.S., Gaudilliere B, & Aghaeepour N. (2020). Integration of mechanistic immunological knowledge into a machine learning pipeline improves predictions. Nature machine intelligence, 2(10), 619-628.

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Single-Cell Metal Barcoding Protocol

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Formalin-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incubated in PBS containing barcoding reagents (102 Pd, 104 Pd, 105 Pd, 106 Pd, 108 Pd, 110 Pd, 113In and 115In) at a final concentration of 50 nM for 30 min at room temperature and then washed three times with CSM²⁹. Barcoded cells were then pooled and stained with the metal-conjugated antibody mix at room temperature for 1 h³⁰. The antibody mix was removed by washing the cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) was diluted in PBS with 1.6% PFA and incubated with the cells at 4 °C overnight. On the day before measurement, the intercalator solution was removed and cells were washed with CSM, PBS, and ddH₂O. After the last washing step, the cells were resuspended in ddH₂O and filtered through a 70-μm strainer.

Kumar S., Lun X.K., Bodenmiller B., Rodríguez Martínez M, & Koeppl H. (2020). Stabilized Reconstruction of Signaling Networks from Single-Cell Cue-Response Data. Scientific Reports, 10, 1233.

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Multiplexed single-cell metal-tag barcoding

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PFA-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incubated in PBS containing barcoding reagents (102Pd, 104Pd, 105Pd, 106Pd, 108Pd, 110Pd, 113In and 115In) at a final concentration of 100 nM for 30 minutes at room temperature and then washed three times with CSM (Bodenmiller et al., 2012) . Barcoded cells were then pooled and stained with the metal-conjugated antibody mix (Supplementary Table S4) at room temperature for 1 hour. The antibody mix was removed by washing cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) diluted in PBS with 1.6% PFA was incubated overnight with the cells at 4 • C. On the day of the measurement, the intercalator solution was removed, and cells were washed with CSM, PBS, and ddH 2 O. After the last washing step, cells were resuspended in ddH 2 O and filtered through a 70-µm strainer.

Wade J.D., Lun X., Bodenmiller B., & Voit E.O. (2020). Multidimensional single-cell modeling of cellular signaling.

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