All procedures were carried out as previously described [2 (link)] in accordance with Home Office Regulations (UK) and the UK Coordinating Committee on Cancer Research guidelines and by locally approved protocols (Home Office Project licence no. 40-3306). In some instances, CDX were passaged following disaggregation using a human tumour dissociation kit (Miltenyi Biotech) following the manufacturer's instructions. Dead cells were removed from the disaggregated tumour with a dead cell removal kit (Miltenyi Biotech) following the manufacturer's instructions. Murine cells were removed by mixing 20 µl anti-mouse IgG2a+b microbeads (Miltenyi Biotech), 10 µl anti-mouse MHC Class I antibody (eBioscience), and 500 µl binding buffer (Miltenyi Biotech), incubating at 4°C for 30 min, mixing with disaggregated tumour cells and incubating at room temperature for 15 min. Cell–bead mixture was then applied to an LS column (Miltenyi Biotech) in a MidiMACS separator (Miltenyi Biotech), the flow through collected, the column washed with 4 × 3 ml binding buffer, and the flow through and wash containing the human cells combined. Disaggregated cells were collected by centrifugation, resuspended in 10% DMSO in fetal bovine serum (Biowest) and stored at −80°C or in liquid nitrogen before re-implantation.
Binding buffer
Manufactured by Miltenyi Biotec
Binding buffer is a solution used in various laboratory techniques, such as affinity chromatography and immunoprecipitation, to facilitate the binding of target molecules to a solid support or matrix. It provides the optimal conditions for the interaction between the target molecule and the binding ligand or antibody. The specific composition and pH of the binding buffer can vary depending on the application and the properties of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Dead cell removal kit, by Miltenyi Biotec (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Lymphoprep, by STEMCELL (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Facs navios, by Beckman Coulter (2 mentions)
Human tumour dissociation kit, by Miltenyi Biotec (1 mentions)
Anti mouse igg2a b microbeads, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Midimacs separator, by Miltenyi Biotec (1 mentions)
Facscanto 2, by BD (1 mentions)
Mitotracker red fm, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Annexin 5 fitc, by Miltenyi Biotec (1 mentions)
Dulbecco s pbs, by Merck Group (1 mentions)
Heat inactivated fcs, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Miltenyi Biotec (1 mentions)
7 protocols using binding buffer
1
Xenograft Tumor Disaggregation and Cell Cryopreservation
2
Apoptosis Quantification in B-ALL Cells
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Apoptotic rate of B-cells was assessed using FITC-Annexin V/Propidium Iodide (PI)
staining, as previously described. Briefly, B-ALL cells were washed twice with PBS
and then stained with APC-conjugated anti-CD19 for 15 minutes in the dark at room
temperature. Cells were re-suspended in binding buffer (MiltenyiBiotec), and
FITC-conjugated Annexin V (MiltenyiBiotec) was added at 1 μg/mL final
concentration. The mixture was incubated at room temperature for 15 minutes in the
dark. Membrane integrity was assessed by PI staining, immediately before flow
cytometric analysis, by using a FACS Canto II (BD Biosciences).
staining, as previously described. Briefly, B-ALL cells were washed twice with PBS
and then stained with APC-conjugated anti-CD19 for 15 minutes in the dark at room
temperature. Cells were re-suspended in binding buffer (MiltenyiBiotec), and
FITC-conjugated Annexin V (MiltenyiBiotec) was added at 1 μg/mL final
concentration. The mixture was incubated at room temperature for 15 minutes in the
dark. Membrane integrity was assessed by PI staining, immediately before flow
cytometric analysis, by using a FACS Canto II (BD Biosciences).
3
Comprehensive Immune Cell Analysis
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Flow cytometry was carried out by using LSR Fortessa X-20 (BD) and cell sorting FACS Aria machines (BD). For apoptosis assay, 4×10 5 neutrophils were stained with AnnexinV-FITC in Binding Buffer (Miltenyi Biotec) and Live/Dead (Zombie Aqua Fixable, BioLegend). For mitochondria analyses, MitoTracker Green and MitoTracker Red FM or MitoSOX Red Mitochondrial Superoxide Indicator, were added to neutrophils for 15 min at RT in MACS buffer or PBS, respectively (ThermoFisher Scientific). Alternatively, neutrophils were incubated with TMRM in RPMI for 20 min at 37°C (Abcam). For phagocytosis assay, 10 5 neutrophils stimulated with zymosan-FITC (50μg/ml, Fluorescein zymosanA BioParticles conjugates, FisherScientific), C. albicans-GFP (MOI1:1) or Escherichia coli-GFP (MOI1:10) for 45 min. Cells were stained with surface antibodies in MACS buffer (online supplemental table S2).
4
Isolation and Enrichment of PBMCs for CITE-seq
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PBMCs were isolated from blood samples using Lymphoprep (StemCell Technologies) density gradient centrifugation according to the manufacturer’s instructions. Single-cell suspensions were then washed with Dulbecco’s PBS (Sigma) and frozen in aliquots containing 5–10 million cells in 90% (vol/vol) heat-inactivated FCS (Gibco) and 10% (vol/vol) DMSO (Sigma-Aldrich). On the day of the experiment, the cells were thawed for 1 min, transferred to wash buffer (PBS supplemented with 2% (vol/vol) FCS and 2 mM EDTA) and centrifuged at 500g for 5 min. Resuspended cells were passed through a 30-μm filter and counted before live-cell magnetic-activated cell sorting (MACS) enrichment with the dead cell removal kit (Miltenyi Biotech), per the manufacturer’s instructions. Cell pellets were resuspended in microbeads and incubated at room temperature for 15 min. Each stained sample was passed through an LS column and rinsed with binding buffer (all from Miltenyi Biotec) before centrifugation. Cell pellets were resuspended in wash buffer and counted for antibody staining by cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).
5
PBMC Isolation and Live Cell Enrichment
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Newcastle:
PBMCs were isolated from blood samples using Lymphoprep (StemCell Technologies) density gradient centrifugation as per manufacturer's instructions. Single cell suspensions were then washed with Dulbecco's phosphate buffered saline (PBS) (Sigma) and frozen in 5-10 million cell aliquots in 90% (v/v) heat inactivated fetal calf serum (FCS) (Gibco) 10% (v/v) DMSO (Sigma Aldrich). On the day of the experiment the cells were thawed for 1 min, transferred to Wash buffer (PBS supplemented with 2% (v/v) FCS and 2 mM EDTA), and centrifuged at 500 g for 5 min.
Resuspended cells were passed through a 30 μm filter and counted prior to live cell MACS enrichment with the Dead cell removal kit (Miltenyi Biotech) as per manufacturer's instructions.
Cell pellets were resuspended in microbeads and incubated at room temperature for 15 min. Each stained sample was passed through an LS column (Miltenyi Biotec) and rinsed with Binding buffer (Miltenyi Biotec) before centrifugation. Cell pellets were resuspended in Wash buffer and counted for CITE-seq antibody staining.
PBMCs were isolated from blood samples using Lymphoprep (StemCell Technologies) density gradient centrifugation as per manufacturer's instructions. Single cell suspensions were then washed with Dulbecco's phosphate buffered saline (PBS) (Sigma) and frozen in 5-10 million cell aliquots in 90% (v/v) heat inactivated fetal calf serum (FCS) (Gibco) 10% (v/v) DMSO (Sigma Aldrich). On the day of the experiment the cells were thawed for 1 min, transferred to Wash buffer (PBS supplemented with 2% (v/v) FCS and 2 mM EDTA), and centrifuged at 500 g for 5 min.
Resuspended cells were passed through a 30 μm filter and counted prior to live cell MACS enrichment with the Dead cell removal kit (Miltenyi Biotech) as per manufacturer's instructions.
Cell pellets were resuspended in microbeads and incubated at room temperature for 15 min. Each stained sample was passed through an LS column (Miltenyi Biotec) and rinsed with Binding buffer (Miltenyi Biotec) before centrifugation. Cell pellets were resuspended in Wash buffer and counted for CITE-seq antibody staining.
6
Apoptosis Detection by Annexin V-FITC
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Apoptotic cells were measured using Annexin V-FITC Apoptosis Detection Kit (Miltenyi Biotec, Gaithersburg, MD, USA), according to the manufacturer’s recommendations. Briefly, cells were seeded in six-well plates and allowed to adhere for 24 h. After plating, the culture medium was replaced by a fresh one either containing effectors or not, and cells were further incubated for 2 days before assay. For the detection of apoptosis, cells were collected, centrifuged, washed, and resuspended in 100 µL 1 × Binding Buffer (Miltenyi Biotec). After the addition of 5 µL annexin-V-FITC, cells were incubated for 20 min and then analyzed by flow cytometry (FACS Beckman Coulter Navios, Brea, CA, USA).
7
Measuring Apoptosis using Annexin V-PE
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Apoptotic cells were measured using Annexin V-PE Apoptosis Detection Kit I (Miltenyi Biotec), according to the manufacturer’s recommendations. Briefly, cells were seeded in the corresponding culture medium. One day after plating, the culture medium was replaced by a fresh one and cells were further incubated for 2 days. For detection of apoptosis, cells were collected, centrifuged, washed and resuspended in 100 μl 1× Binding Buffer (Miltenyi Biotec). After addition of 5 μl annexin V-PE, cells were incubated for 15 minutes and then analyzed by flow cytometry (FACS Beckman Coulter Navios).
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