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Maxpal fix and pem buffer

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MaxPal Fix is a fixation reagent used to preserve cellular structure and protein content for downstream applications. Pem Buffer is a permeabilization and blocking buffer used to prepare samples for immunofluorescence analysis.

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Lab products found in correlation

Eq calibration beads, by Standard BioTools (5 mentions) Human fc receptor blocking solution, by BioLegend (5 mentions) Maxpal cell staining buffer, by Standard BioTools (5 mentions) Cell id cisplatin, by Standard BioTools (5 mentions) Cytof2 instrument, by Standard BioTools (3 mentions) Cytof2, by Standard BioTools (1 mentions) Helios cytof mass cytometer, by Standard BioTools (1 mentions)

5 protocols using maxpal fix and pem buffer

1

CyTOF Assay for Cell Phenotyping

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The CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3 × 106 cells were stained with 5 mM Cell-ID™ Cisplatin (Fluidigm, San Francisco, CA) for 5min and quenched with MaxPal Cell Staining Buffer (Fluidigm) using 5 times the volume of the cell suspension. After centrifugation, cell suspensions (50 μl) were incubated with 5 μL of human Fc-receptor Blocking solution (Biolegend, San Diego, CA) for 10min and 50 μL of pre-mixed antibody cocktail (Table S2) for 30 min. After washing, cells were incubated with 1ml of cell intercalation solution (125nM MaxPal Intercalator-Ir into 1ml MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a CyTOF II instrument (Fluidigm).
Yang Z.Z., Jin Kim H., Villasboas J.C., Price-Troska T., Jalali S., Wu H., Luchtel R.A., Polley M.Y., Novak A.J, & Ansell S.M. (2019). Mass Cytometry Analysis Reveals that Specific Intratumoral CD4+ T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma. Cell reports, 26(8), 2178-2193.e3.
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2

CyTOF Immunophenotyping of Cell Samples

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The CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3×106 cells were stained with 5 µM Cell-IDTM Cisplatin (Fluidigm, San Francisco, California, USA) for 5 min and quenched with MaxPal Cell Staining Buffer (Fluidigm) using five times the volume of the cell suspension. After centrifugation, cell suspensions (50 µL) were incubated with 5 µL of human Fc-receptor Blocking solution (BioLegend, San Diego, California, USA) for 10 min and 50 µL of pre-mixed antibody cocktail for 30 min. For the staining antibody panel, refer to this publication.20 (link) After washing, cells were incubated with 1 mL of cell intercalation solution (125 nM MaxPal Intercalator-Ir into 1 mL MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a CyTOF II instrument (Fluidigm).
Wu H., Tang X., Kim H.J., Jalali S., Pritchett J.C., Villasboas J.C., Novak A.J., Yang Z.Z, & Ansell S.M. (2021). Expression of KLRG1 and CD127 defines distinct CD8+ subsets that differentially impact patient outcome in follicular lymphoma. Journal for Immunotherapy of Cancer, 9(7), e002662.
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3

CyTOF Assay for Cell Phenotyping

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The CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3 × 106 cells were stained with 5 mM Cell-ID™ Cisplatin (Fluidigm, San Francisco, CA) for 5min and quenched with MaxPal Cell Staining Buffer (Fluidigm) using 5 times the volume of the cell suspension. After centrifugation, cell suspensions (50 μl) were incubated with 5 μL of human Fc-receptor Blocking solution (Biolegend, San Diego, CA) for 10min and 50 μL of pre-mixed antibody cocktail (Table S2) for 30 min. After washing, cells were incubated with 1ml of cell intercalation solution (125nM MaxPal Intercalator-Ir into 1ml MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a CyTOF II instrument (Fluidigm).
Yang Z.Z., Jin Kim H., Villasboas J.C., Price-Troska T., Jalali S., Wu H., Luchtel R.A., Polley M.Y., Novak A.J, & Ansell S.M. (2019). Mass Cytometry Analysis Reveals that Specific Intratumoral CD4+ T Cell Subsets Correlate with Patient Survival in Follicular Lymphoma. Cell reports, 26(8), 2178-2193.e3.
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4

CyTOF Cellular Staining and Analysis

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Metal-conjugated antibodies used for CyTOF analyses are presented in Key resource table. The CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3 × 106 cells were stained with 5mM Cell-ID Cisplatin (Fluidigm, San Francisco, CA) for 5 minutes and quenched with MaxPal Cell Staining Buffer (Fluidigm) using 5 times the volume of the cell suspension. After centrifugation, cell suspensions (50 μl) were incubated with 5 μl of human Fc-receptor blocking solution (Biolegend, San Diego, CA) for 10 minutes and 50 μl of pre-mixed antibody cocktail for 30 minutes. After washing, cells were incubated with 1 ml of cell intercalation solution (125nM MaxPal Intercalator-Ir into 1 ml MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a Helios CyTOF mass cytometer (Fluidigm).
Hailemichael Y., Johnson D.H., Abdel-Wahab N., Foo W.C., Bentebibel S.E., Daher M., Haymaker C., Wani K., Saberian C., Ogata D., Kim S.T., Nurieva R., Lazar A.J., Abu-Sbeih H., Fa-ak F., Matthew A., Wang Y., Falohun A., Trinh V., Zobniw C., Spillson C., Burks J.K., Awiwi M., Elsayes K., Soto L.S., Melendez B.D., Davies M., Wargo J., Curry J., Yee C., Lizee G.A., Singh S., Sharma P., Allison J.P., Hwu P., Ekmekcioglu S, & Diab A. (2022). Interleukin-6 blockade abrogates immunotherapy toxicity and promotes tumor immunity. Cancer cell, 40(5), 509-523.e6.
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5

Comprehensive CyTOF Immune Cell Profiling

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CyTOF assay was performed according to the manufacturer’s instruction. Briefly, 3×106 cells were stained with 5μM Cell-ID™ Cisplatin (Fluidigm, San Francisco, CA) for 5min and quenched with MaxPal Cell Staining Buffer (Fluidigm) using 5× the volume of the cell suspension. After centrifugation, cell suspensions (50μl) were incubated with 5μl of human Fc-receptor Blocking solution (Biolegend, San Diego, CA) for 10min and 50μl of pre-mixed antibody cocktail for 30 min. After washing, cells were incubated with 1ml of cell intercalation solution (125 nM MaxPal Intercalator-Ir into 1ml MaxPal Fix and Pem Buffer, Fluidigm) overnight at 4°C. Cells were centrifuged with MaxPal Water and pelleted. The pelleted cells were suspended with EQ Calibration Beads (Fluidigm) and cell events were acquired by a CyTOF II instrument (Fluidigm). The data were analyzed using online software Cytobank, viSNE map and SPADE.
Yang Z.Z., Kim H.J., Villasboas J.C., Chen Y.P., Price-Troska T., Jalali S., Wilson M., Novak A.J, & Ansell S.M. (2017). Expression of LAG-3 defines exhaustion of intratumoral PD-1+ T cells and correlates with poor outcome in follicular lymphoma. Oncotarget, 8(37), 61425-61439.
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