Prior to antibody staining, mass tag cellular barcoding of prepared samples was performed by incubating cells with distinct combinations of isotopically-purified palladium ions chelated by isothiocyanobenzyl-EDTA as previously described ((Zunder et al. 2015 (link))). After counting, 1*106 cells from aliquot were barcoded with distinct combinations of stable Pd isotopes for 15 min at room temperature on a shaker in Maxpar Barcode Perm Buffer (Fluidigm, cat#201057). Cells were washed twice with cell staining media (PBS with 0.5% BSA and 0.02% NaN3), and pooled into a single 15 ml tube.
Maxpar barcode perm buffer
Manufactured by Standard BioTools
Sourced in United States
The Maxpar Barcode Perm Buffer is a reagent designed for the permeabilization and fixation of cells in preparation for mass cytometry (CyTOF) analysis. It is used to allow antibody-based staining of intracellular proteins and targets.
Automatically generated - may contain errors
Lab products found in correlation
Cell id 20 plex pd barcoding kit, by Standard BioTools (3 mentions)
Cytof2 instrument, by Standard BioTools (1 mentions)
Eq four element calibration beads, by Standard BioTools (1 mentions)
Cytof2 mass cytometer, by Standard BioTools (1 mentions)
Cell id cisplatin, by Standard BioTools (1 mentions)
Cell staining buffer, by Standard BioTools (1 mentions)
5 protocols using maxpar barcode perm buffer
1
Mass Tag Cellular Barcoding for Samples
2
Cellular Barcoding for Mass Cytometry
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Prior to antibody staining, mass tag cellular barcoding of prepared samples was performed by incubating cells with distinct combinations of isotopically-purified palladium ions chelated by isothiocyanobenzyl-EDTA as previously described ((Zunder et al., 2015 (link))). After counting, 1∗106 cells from aliquot were barcoded with distinct combinations of stable Pd isotopes for 15 min at room temperature on a shaker in Maxpar Barcode Perm Buffer (Fluidigm, cat#201057). Cells were washed twice with cell staining media (PBS with 0.5% BSA and 0.02% NaN3), and pooled into a single 15 mL tube.
3
CyTOF Barcoded Sample Batch Processing and Analysis
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Barcoded sample batches were processed simultaneously to reduce data collection variability. Sample barcoding and data acquisition were carried out as previously described (18 (link)). Briefly, cells were washed twice in Maxpar® Barcode perm buffer (Fluidigm; US) and each sample was stained with a different barcode combination using Cell-ID 20-plex Pd-barcoding kit (Fluidigm; US) for 30 min at room temperature. After washing twice in CSB, samples were pooled together into one tube. Cells were incubated for 20 min at room temperature with 125 nM Cell-ID™ DNA Intercalator-Ir191/193 (Fluidigm; US), washed twice with CSB and once with Di water and lastly resuspended with 0.1 % EQ four element calibration beads (Fluidigm; US). Data were acquired with a CyTOF2® instrument (Fluidigm; US). After acquisition, data were concatenated, normalized using mass bead signal and de-barcoded using the CyTOF 2 software.
4
Mass Cytometry Barcoding for Batch Processing
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To reduce data collection variability, samples were processed in batches of 5, including similar number of samples from each group, and barcoded with combinations of three unique palladium isotopes (26 (link)). Barcoding was performed after antibody staining, as PFA-based fixation used during barcoding resulted in drastic reduction in transcription factor staining when used before the FOXP3 fixation/permeabilization buffer (Supplementary Figure 1 ).
Cells were barcoded using Cell-ID 20-plex Pd-barcoding kit (Fluidigm) according to the manufacturer's instructions. Briefly, cells were washed twice with Maxpar barcode perm buffer (Fluidigm), and a different barcode set was added to each sample for 30 min at room temperature. After washing the samples twice with CSM, all samples were combined into one tube. Finally, cells were stained with 125 nM Ir191/193 DNA intercalator (Cell-ID Intercalator-Ir, Fluidigm) for 20 min, washed in Di water, filtered through a 35 μm nylon mesh and resuspended to 0.5 × 106 cells/ml with 0.1% EQ four element calibration beads (Fludigm). Data acquisition was done with a CyTOF 2 mass cytometer (Fluidigm) at an event rate of 300–500 cells/s. After data acquisition,.fcs files were concatenated, normalized using mass bead signal (27 (link)) and debarcoded using the CyTOF 2 software prior to analysis.
Cells were barcoded using Cell-ID 20-plex Pd-barcoding kit (Fluidigm) according to the manufacturer's instructions. Briefly, cells were washed twice with Maxpar barcode perm buffer (Fluidigm), and a different barcode set was added to each sample for 30 min at room temperature. After washing the samples twice with CSM, all samples were combined into one tube. Finally, cells were stained with 125 nM Ir191/193 DNA intercalator (Cell-ID Intercalator-Ir, Fluidigm) for 20 min, washed in Di water, filtered through a 35 μm nylon mesh and resuspended to 0.5 × 106 cells/ml with 0.1% EQ four element calibration beads (Fludigm). Data acquisition was done with a CyTOF 2 mass cytometer (Fluidigm) at an event rate of 300–500 cells/s. After data acquisition,.fcs files were concatenated, normalized using mass bead signal (27 (link)) and debarcoded using the CyTOF 2 software prior to analysis.
5
CyTOF Barcoding of Cell Samples
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For all CyTOF experiments, the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm) was used following the manufactural instructions. In short, 1 million cells per condition and per line were washed with PBS and then incubated with Cell-ID Cisplatin (Fluidigm) for 10 min at RT. Afterwards, cells were fixed with MaxPar Fix Buffer (Fluidigm) for 10 min at RT, washed with MaxPar Barcode Perm Buffer (Fluidigm), and incubated with the appropriate barcode for 30 min at RT. Finally, cells were washed with Cell Staining Buffer (Fluidigm) and combined depending on the CyTOF experiment in one or more tubes before antibody staining. Depending on the planned CyTOF experiment, a specific barcoding strategy was developed in order to minimize technical bias and highlight biological differences.
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