All the kidney samples were processed in the same batch. CyTOF analysis was performed by PLTTech, Inc. (Hangzhou, China) according to a published protocol [12 (link)]. The kidney tissue was dissociated into a single-cell suspension with a mixture of DNase I, collagenase IV, and hyaluronidase (Sigma-Aldrich). Immune cells were enriched using Percoll density gradient media (Sigma-Aldrich), and erythrocytes were fully removed using ACK Lysing Buffer (Sigma-Aldrich). Qualified samples were blocked and stained with a mixed panel of surface antibodies, followed by overnight fixation. After fixing with Fix & Perm Buffer (Fluidigm), the cells were incubated in an intracellular antibody mix. The signals of the stained cells were detected using a CyTOF system (Fluidigm). The types of immune cells were identified via nonlinear dimensionality reduction (t-SNE), followed by density clustering.
Percoll density gradient media
Manufactured by Merck Group
Percoll density gradient media is a colloidal silica-based solution used for the separation and purification of cells, organelles, and other biological particles. It provides a versatile and gentle method for density gradient centrifugation, allowing the effective separation of different cell types or subcellular components based on their density.
Automatically generated - may contain errors
Lab products found in correlation
Ack lysing buffer, by Merck Group (3 mentions)
Collagenase 4, by Merck Group (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Cytof system, by Standard BioTools (2 mentions)
Dnaase, by Merck Group (2 mentions)
Fix perm buffer, by Standard BioTools (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Hyaluronidase type 5, by Merck Group (1 mentions)
Doxycycline hyclate, by Merck Group (1 mentions)
Dnase type 4, by Merck Group (1 mentions)
Human il 2, by BioLegend (1 mentions)
Murine il 2, by BioLegend (1 mentions)
Murine ifn γ, by Abcam (1 mentions)
Anti pd 1, by BioXCell (1 mentions)
Human ifn γ, by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Rat igg2a, by BioXCell (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
4 protocols using percoll density gradient media
1
Comprehensive Kidney Immune Profiling
2
Cytokine and Antibody Modulation in Vivo
3
Comprehensive CyTOF Immune Profiling of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
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CyTOF analyses were performed by PLTTech Inc. (Hangzhou, China) according to the previously described protocol.11 (link) In brief, tumour tissue was dissociated into single cells with DNAase, collagenase IV and hyaluronidase (Sigma-Aldrich, Saint Louis, MO, USA). Immune cells were enriched using Percoll density gradient media (Sigma-Aldrich), and red blood cells were removed using ACK Lysing Buffer (Sigma-Aldrich). Qualified samples were blocked and stained for 30 min with a surface antibody mix panel developed in-house (online table S1 ), followed by fixation overnight. Permeabilization buffer was applied, and the cells were incubated in an intracellular antibody mix. The cells were rinsed, and the signals were detected using a CyTOF system (Helios, Fluidigm, South San Francisco, CA, USA). The types of immune cells were identified via nonlinear dimensionality reduction [t-distributed stochastic neighbour embedding (tSNE)], followed by density clustering.12 (link)
4
CyTOF Profiling of Mouse Liver Immune Cells
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CyTOF analysis was performed by PLTTech Inc. (Hangzhou, China) according to the protocol described previously.
33 (link) In brief, mouse liver tissue was dissociated into single cells with DNAase, collagenase IV, and hyaluronidase (Sigma‐Aldrich, Saint Louis, MO, USA). Immune cells were concentrated using Percoll density gradient media (Sigma‐Aldrich), while red blood cells were removed using ACK Lysing Buffer (Sigma‐Aldrich). Qualified samples were gathered in blocks and stained for 30 min with a panel of 42 antibodies, followed by overnight fixation. Permeabilisation buffer was applied, and the cells were incubated in an intracellular antibody mixture. The cells were rinsed, and the signals were detected using a CyTOF system (Helios, Fluidigm, South San Francisco, CA, USA). The types of immune cells were identified via nonlinear dimensionality reduction (viSNE) followed by density‐based clustering.
33 (link) In brief, mouse liver tissue was dissociated into single cells with DNAase, collagenase IV, and hyaluronidase (Sigma‐Aldrich, Saint Louis, MO, USA). Immune cells were concentrated using Percoll density gradient media (Sigma‐Aldrich), while red blood cells were removed using ACK Lysing Buffer (Sigma‐Aldrich). Qualified samples were gathered in blocks and stained for 30 min with a panel of 42 antibodies, followed by overnight fixation. Permeabilisation buffer was applied, and the cells were incubated in an intracellular antibody mixture. The cells were rinsed, and the signals were detected using a CyTOF system (Helios, Fluidigm, South San Francisco, CA, USA). The types of immune cells were identified via nonlinear dimensionality reduction (viSNE) followed by density‐based clustering.
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