Maxpar cell staining buffer

Manufactured by Standard BioTools

Sourced in United States, Cameroon

The Maxpar Cell Staining Buffer is a laboratory reagent designed to prepare cells for flow cytometry analysis using Maxpar technology. It is a buffer solution that maintains the integrity and viability of cells during the staining process, ensuring accurate and reliable results.

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Cell Staining Buffer (14 citations) MaxPar Cell Staining Buffer (CSB, (5 citations) Staining buffer (4 citations) Maxpar staining buffer (3 citations) Maxpar Cell Staining Buffer (MCSB; (2 citations)

41 protocols using maxpar cell staining buffer

Immune Cell Profiling of TBI Mice

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Immune cells were isolated from the sites of injury in the TBI mice and prepared for cell-staining protocols by Fluidigm. Briefly, cells were stained with 0.5 μM cell-ID cisplatin (Fluidigm, catalog no. 201064) for 2 min, followed by adding 2 ml of MaxPar Cell Staining Buffer (Fluidigm, catalog no. 201068) to stop the reaction. After centrifugation and disposal of the supernatant, the cells were resuspended in MaxPar Cell Staining Buffer to a volume of 50 μl. Fifty microliters of a metal-conjugated surface-marker antibody cocktail was added, and the samples were incubated for 30 min at room temperature. For intracellular staining, cells were fixed in 1 ml of 1× MaxPar Fix I Buffer (Fluidigm, catalog no. 201065) at room temperature for 20 min. After washing twice with 2 ml of MaxPar Perm-S Buffer (Fluidigm, catalog no. 201066), the cells were incubated with 50 μl of an intracellular antibody cocktail for 30 min. After washing twice with 2 ml of MaxPar Cell Staining Buffer, the cells were resuspended in 1 ml of the intercalation solution and incubated overnight at 4°C. After washing twice with 2 ml of MaxPar Cell Staining Buffer, the cell concentration was adjusted to 2.5 to 5 × 10⁵/ml with MaxPar Water (Fluidigm, catalog no. 201069) for the CyTOF assay.

Li Z., Xiao J., Xu X., Li W., Zhong R., Qi L., Chen J., Cui G., Wang S., Zheng Y., Qiu Y., Li S., Zhou X., Lu Y., Lyu J., Zhou B., Zhou J., Jing N., Wei B., Hu J, & Wang H. (2021). M-CSF, IL-6, and TGF-β promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery. Science Advances, 7(11), eabb6260.

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Spleen Cell Isolation and Enrichment

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Spleen was homogenized, passed through 70 μm filters, washed, and subjected to red blood cell lysis (ACK lysing buffer, GIBCO). The red blood cell lysis was stopped by adding washing buffer (MaxPar Cell staining Buffer, Fluidigm), and the homogenate was then passed through 30 μm filters and washed again before final suspension in MaxPar Cell staining Buffer. The cell suspension was adjusted to 2x10⁶ cells/500 μl.

Sbierski-Kind J., Grenkowitz S., Schlickeiser S., Sandforth A., Friedrich M., Kunkel D., Glauben R., Brachs S., Mai K., Thürmer A., Radonić A., Drechsel O., Turnbaugh P.J., Bisanz J.E., Volk H.D., Spranger J, & von Schwartzenberg R.J. (2022). Effects of caloric restriction on the gut microbiome are linked with immune senescence. Microbiome, 10, 57.

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Mass Cytometry Immunophenotyping Protocol

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Mass cytometry staining and analyses were performed as described previously [25 (link)]. The antibodies used for the mass cytometry analysis are summarized in Table S1. PBMCs were stained after washing with phosphate-buffered saline (PBS) supplemented with 2% fetal calf serum (FCS). Cells were incubated in 5 μM Cell-ID Cisplatin solution (Fluidigm, CA, USA) in PBS, washed with MaxPar Cell Staining Buffer (Fluidigm), and stained with a mixture of surface antibodies. After washing, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. The fixed and permeabilized cells were stained with intracellular antibodies. After washing twice, cells were rested in 125 nM MaxPar Intercalator-Ir (Fluidigm) diluted with 2% paraformaldehyde in PBS at 4 °C for 60 min. Cells were then washed twice with MaxPar Cell Staining Buffer and once with MaxPar water (Fluidigm), distilled water with minimal heavy-element contamination to reduce the background level. Cells suspended in MaxPar water supplemented with 10% EQ Four Element Calibration Beads (Fluidigm) were applied to the Helios system (Fluidigm), and data were acquired at a speed below 300 events/s. Data analyses were performed using FlowJo v10.7 (BD Biosciences, Franklin Lakes, CA, USA) as described previously [26 (link)].

Fujioka Y., Sugiyama D., Matsumura I., Minami Y., Miura M., Atsuta Y., Ohtake S., Kiyoi H., Miyazaki Y., Nishikawa H, & Takahashi N. (2021). Regulatory T Cell as a Biomarker of Treatment-Free Remission in Patients with Chronic Myeloid Leukemia. Cancers, 13(23), 5904.

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Multiparameter Analysis of Murine Lung Cells

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Low-density cells (3 × 10⁶) isolated from perfused lungs of C57BL/6 mice were incubated for 5 min with 1 μM of Cell-ID Cisplatin (Fluidigm) at room temperature to label dead cells, then washed with Maxpar Cell Staining Buffer (Fluidigm). To block non-specific Ab binding, cells were incubated with anti-mouse CD16/CD32 Abs, normal mouse serum, and rat serum, then incubated with 50 μL of metal-conjugated Abs (Supplementary Table 1) for 30 min. Staining of cells with fluoresce-conjugated primary Abs was followed by metal-conjugated secondary Abs (Supplementary Table 1). Cells were fixed in 2% paraformaldehyde for 60 min, washed with Maxpar Cell Staining Buffer, and then incubated in 125 nM Cell-ID Intercalator-Ir (Fluidigm) in Maxpar Fix and Perm Buffer (Fluidigm) overnight. On the following day, cells were washed and filtered through BelArt SP Flowmi cell strainers (Fisher Scientific), and analyzed on a Helios mass cytometer (Fluidigm) with CyTOF 6.7 software (Fluidigm). Data were analyzed using Cytobank platform (Cytobank).

Izumi G., Nakano H., Nakano K., Whitehead G.S., Grimm S.A., Fessler M.B., Karmaus P.W, & Cook D.N. (2021). CD11b+ lung dendritic cells at different stages of maturation induce Th17 or Th2 differentiation. Nature Communications, 12, 5029.

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Multiplexed Single-Cell CyTOF Analysis

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Antibodies were purchased pre-conjugated when commercially available. Non-available antibodies were conjugated to lanthanide metals using Maxpar X8 antibody labeling kit according to the manufacturer protocol (version 10). The antibody cocktail used is listed in Table S7. For barcoding and staining, cells were washed with Maxpar PBS buffer (Fluidigm #201058) and stained with 0.5 μM Cell-ID Cisplatin Viability Stain (Fluidigm #201064) in 200 μL Maxpar PBS for 5 mintutes at room temperature for dead cell exclusion. The reaction was quenched with Maxpar Cell Staining Buffer (CSB, Fluidigm #201063) and cells fixed, permeabilized and barcoded using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm #201060) as per the manufacturers user guide. Barcoded cells were washed, combined and stained with the antibody cocktail as per Table S7 for 30 minutes at room temperature. Cells were washed with Maxpar Cell Staining Buffer (Fluidigm #201068), fixed in 1.6% formaldehyde, washed and resuspended in Fix&Perm Buffer (Fluidigm Cat#201067) with Cell-ID intercalator-Ir (Fluidigm #201103B) and incubated overnight at 4°C. The following day, cells were washed and analyzed on a Helios (Fluidigm). The mass cytometer was tuned and QC was run prior to acquiring samples according to the manufacturers’ recommendations.

Psaila B., Wang G., Rodriguez-Meira A., Li R., Heuston E.F., Murphy L., Yee D., Hitchcock I.S., Sousos N., O’Sullivan J., Anderson S., Senis Y.A., Weinberg O.K., Calicchio M.L., Iskander D., Royston D., Milojkovic D., Roberts I., Bodine D.M., Thongjuea S, & Mead A.J. (2020). Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets. Molecular Cell, 78(3), 477-492.e8.

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Multiparameter Profiling of Immune Cells

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Cryopreserved LPMC and PBMC were barcoded with a combination of anti-CD45 antibodies (89Y, 115In, and 175Lu) for 30 min at RT and washed with Maxpar Cell Staining Buffer (Fluidigm). After centrifugation, cells were stained with an antibody mix (SI Appendix, Tables S1 and S5) for 45 min at RT. After washes, cells were incubated with cisplatin (1:1,000, Cell-ID™ Cisplatin-¹⁹⁸Pt; Fluidigm) for 5 min at RT, quenched with Maxpar Cell Staining Buffer, and fixed with 2% PFA for 30 min at 4 °C. For intracellular staining, eBioScience Fix/Perm Buffer (Invitrogen) was added to cells and centrifuged, stained with markers dissolved in Fix/Perm Buffer for 45 min at 4 °C, and washed with Fix/Perm Buffer. After centrifugation, the pellet was incubated with iridium (1:1,000, Cell-ID™ Intracalator-Ir; Fluidigm) overnight at 4 °C, washed, and acquired on CyTOF Helios (Fluidigm). EQ beads were used for normalization.

Yokoi T., Murakami M., Kihara T., Seno S., Arase M., Wing J.B., Søndergaard J.N., Kuwahara R., Minagawa T., Oguro-Igashira E., Motooka D., Okuzaki D., Mori R., Ikeda A., Sekido Y., Amano T., Iijima H., Ozono K., Mizushima T., Hirota S., Ikeuchi H, & Takeda K. (2022). Identification of a unique subset of tissue-resident memory CD4+ T cells in Crohn’s disease. Proceedings of the National Academy of Sciences of the United States of America, 120(1), e2204269120.

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CyTOF2 Mass Cytometry Sample Preparation

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Sample preparation. Cryopreserved PBMC were thawed, resuspended in phosphate buffer saline (PBS) to a concentration of 1-3x10⁶ cells/mL and incubated 10 min with FcR Block Reagent (Miltenyi Biotec) prior to Ab staining.
Staining procedure. Purified antibodies were either purchased pre-conjugated from the manufacturer (Fluidigm, San Francisco, CA) or conjugated in-house with the appropriate metal isotope using MaxPar Metal labeling kits (Fluidigm) according to manufacturer’s instructions. For staining, 1x10⁶ cells were washed in Maxpar cell staining buffer (Fluidigm) and stained in 100 μL Maxpar cell staining buffer (Fluidigm) containing the cocktail of 24 Abs listed in Supplemental Table 1. Stained cells were then incubated for 10 min in 1.6% paraformaldehyde (Sigma). DNA staining was performed by overnight incubation in 2 mL of 125 nM Cell-ID Iridium intercalator solution (Fluidigm) at 4°C. Cells were then washed, pelleted, and kept at 4°C until acquisition.
Data Acquisition. Samples were analyzed on a CyTOF2 mass cytometer upgraded to Helios (Fluidigm). Cells were resuspended in EQ™ Four Element Calibration Beads (Fluidigm) diluted to 0.5X in Maxpar ultra-pure water (Fluidigm) and filtered twice through a 50 µm nylon mesh to reach an acquisition rate of 200-500 events per second.

Andrieu T., Mondière P., Jouve P.E., Dussurgey S., Malassigné V., Servanton H., Baseggio L., Davi F., Michallet A.S, & Defrance T. (2022). Mass cytometry analysis reveals attrition of naïve and anergized self-reactive non-malignant B cells in chronic lymphocytic leukemia patients. Frontiers in Oncology, 12, 1020740.

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Mass Cytometry Sample Preparation and Analysis

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Stained cell samples were centrifuged (500 × g, 5 min), and the excess Intercalator-Ir was removed in the supernatant. The cells were resuspended in Maxpar cell staining buffer (Fluidigm) and centrifuged twice to remove all salts. Aliquots of each sample were applied to a hemocytometer to determine cell counts. The samples were washed once in MilliQ H₂O and centrifuged, and the supernatant was removed as completely as possible. Pellets were then kept on ice until immediately prior to analysis.
Calibration bead solution (Fluidigm) was run on the CyTOF2 mass cytometer (Fluidigm) to calibrate it. Each sample was resuspended in a 1:10 dilution of the bead solution to a concentration of 2.5–5.0 × 10⁵ cells/mL and filtered immediately before injection into the instrument. The instrument was thoroughly washed with deionized water between each sample. Results were analyzed using FlowJo software. Each sample was gated for single cells according to DNA content (DNA-Ir), and bead-cell doublets were removed. Background signal was determined by comparison with negative controls. Negative controls included staining in the absence of antigen (such as BrdU antibody staining on cells that were not pulsed with BrdU), and no antibody controls. Gating of positive signal was based on negative control staining in absence of antigen.

Shaklee J., Srivastava K., Brown H., Arriaga E.A., Pierre V.C, & van Berlo J.H. (2018). Development of a Click-Chemistry Reagent Compatible with Mass Cytometry. Scientific Reports, 8, 6657.

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Comprehensive CyTOF Immunophenotyping of Patient-Derived Cells

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Cells from patients in the phase II pilot study were counted and viability was measured using trypan blue exclusion. Two hundred thousand cells from each donor were aliquoted into 5 mL polystyrene U-bottom tubes for barcoding and cytometry by time of flight (CyTOF) staining. Cells were stained with Cisplatin (Fluidigm Product# 201064) followed by barcoding using the Cell-ID 20-Plex Pd Barcoding Kit (FluidigmProduct# 201060). After barcoding, all cells were combined into a single 5 mL polystyrene U-bottom tube and incubated in the surface marker antibody cocktail (Supplementary Table S1) for 30 minutes at 4°C. Following surface staining, cells were then fixed using 2% paraformaldehyde. For intracellular staining, cells were permeabilized by incubation with Triton X 0.1% for 5 minutes at room temperature, followed by incubation with intracellular antibody cocktail (Supplementary Table S1) for 30 minutes at 4°C. Stained cells were then incubated overnight with Cell-ID Intercalator (Fluidigm Product# 201192A). The following morning cells were washed and run on the CyTOF 2 instrument (Fluidigm). Wash steps were completed using either Maxpar PBS (Fluidigm Product# 201058), Maxpar Cell Staining Buffer (Fluidigm Product# 201068), or Millipure Water at 1600 RPM for 4 minutes. Data were visualized in viSNE (Cytobank Inc. RRID:SCR_014043).

Felices M., Wesley E., Bendzick L.E., Kodal B., Hopps R., Grzywacz B., Hinderlie P., Miller J.S, & Geller M.A. (2023). Reverse Translation Identifies the Synergistic Role of Immune Checkpoint Blockade and IL15 to Enhance Immunotherapy of Ovarian Cancer. Cancer Immunology Research, 11(5), 674-686.

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Multi-Dimensional Immunophenotyping of Skin Cells

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The metal-tagged antibodies and reagents were all purchased from Fluidigm, and staining panels were designed using Maxpar Panel Designer. For samples preparation, cells from epidermis and dermis were suspended and washed in serum free RPMI 1640 (11875-093, Gibco), and single cell solution was harvested through a 70 μm cell strainer. All samples were checked for viability by staining with 5 uM cisplatin (Fluidigm) in serum-free RPMI 1640 for 10 min, then washed and centrifuged at 300 × g for 5 min at room temperature (RT). For surface antibodies staining, samples were stained at RT for 20 min and washed with Maxpar Cell staining buffer (Fluidigm). For checking on intracellular cytokines, cells were cultured with or without Cell Activation Cocktail with Brefeldin A for 4 h, then collected cells were stained with surface antibodies and washed twice. Cells were fixed with 1 mL of 1 × Maxpar Fix I buffer at RT for 30 min and then washed twice with 2 mL of 1 × Maxpar Perm-S buffer at room temperature for 5 min at 500 × g, cells were stained with intracellular staining antibodies in 1 × Maxpar Perm-S buffer at room temperature for 30 min. Finally, cells were fixed at RT for 10 min using 1.6% formaldehyde and washed at 800 × g for 10 min, then removed the fix buffer and incubated cell pellet overnight in 125 nM of IntercalatorIridium (Fluidigm) at 4°C.

Tamaru Y, & Doi R.H. (1999). Three Surface Layer Homology Domains at the N Terminus of the Clostridium cellulovorans Major Cellulosomal Subunit EngE. Journal of Bacteriology, 181(10), 3270-3276.

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