Uptake of glucose and palmitate were assessed with fluorescent analogues 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG, Thermo Fisher Scientific, Waltham, MA, USA, N13195), Bodipy FL C12 (D3822) and Bodipy FL C16 (D3821) (Thermo Fisher Scientific, Waltham, MA, USA) using flow cytometry (MACS Quant, Miltenyi Biotec, Bergisch Gladbach, Germany). Here, 20 nM 2-NBDG and 40 nM Bodipy FL C12 or C16 (in PBS) was added to 1e6 lymphoma cells or 95% pure CD43-, B220 + primary B cells for 15 min at 5% CO2 at 37 °C. eFluor 450 (1:2000, Thermo Fisher, Waltham, MA, USA, 65-0863-14) viability dye was used to determine viable cells. Cells were washed before measurement by FACS (MACS Quant, Miltenyi Biotec, Bergisch Gladbach, Germany).
Macsquant
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, United Kingdom, France
The MACSQuant is a flow cytometry system manufactured by Miltenyi Biotec. It is designed to perform automated cell analysis and cell sorting. The system utilizes magnetic cell separation technology to isolate and analyze specific cell populations from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse, by BD (9 mentions)
Facsaria, by BD (9 mentions)
Macsquantify software, by Miltenyi Biotec (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Facscalibur, by BD (6 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions)
Cytofix cytoperm kit, by BD (5 mentions)
Brefeldin a, by BD (4 mentions)
7 aad, by BioLegend (4 mentions)
Flowlogic software, by Miltenyi Biotec (4 mentions)
Anti cd83 apc, by Miltenyi Biotec (4 mentions)
Lsrfortessa, by BD (4 mentions)
Cytofix cytoperm, by BD (4 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (4 mentions)
Attune nxt, by Thermo Fisher Scientific (4 mentions)
Fixation buffer, by BioLegend (3 mentions)
Annexin 5 apc 7aad iodide detection kit, by BD (3 mentions)
Fixable violet dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Anti cd86 apc, by Miltenyi Biotec (3 mentions)
Anti hla abc apc, by Miltenyi Biotec (3 mentions)
374 protocols using macsquant
1
Quantifying Cellular Glucose and Lipid Uptake
2
Assaying SABR signaling in co-culture
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For co-culture assays to test SABR signaling, 1-5×104 transduced NFAT-GFP-Jurkat cells were incubated with equal number of transduced Jurkat cells on 96 well flat or round bottom plates for 8-10 hours. The cells were then acquired on MACSQuant (Miltenyi) or stained with anti-CD69-APC-Cy7 (Biolegend) and then acquired on MACSQuant (Miltenyi). For SABR library assays, 1.5×106 SABR library cells were incubated with 1.5×106Jurkat cells in each well of a 6 well plate. At 8-10 hours after co-culture, cells were harvested, stained with anti-CD69-APC-Cy7 (Biolegend), and sorted on a BD FACS SORP (Becton-Dickinson). Cytotoxicity assays were performed using target cells labeled with CFSE (Biolegend) as described previously19 (link). For empty SABR assays, transduced NFAT-GFP-Jurkat cells were incubated with 100 μg/ml of soluble peptide for 2 hours at 37 °C. Equal numbers of transduced Jurkat cells were then added to the cells, followed by 8-10 hours of co-culture. Gating strategies for these assays is shown in supplementary Fig 9 . Functional avidity assays were performed using KK10 variant peptides as described previously19 (link).
3
Assaying SABR signaling in co-culture
4
Proliferation and Apoptosis Assay in PECs
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Proliferation and apoptosis were assessed in the PECs from both groups (n = 7 in each group; each condition was analyzed in triplicate). The cells were seeded at a density of 5 £ 10 3 cells per well. After 24 hours of starvation (1% serum), the cells were treated for 24 hours with two doses (50 and 500 nM) of I1 or I2 or 5% FCS. Cell growth was measured using the DELFIA Cell Proliferation Kit (PerkinElmer, Courtaboeuf, France) and a time-resolved fluorometer EnVisionTM multilabel reader (PerkinElmer). The same protocol was also performed, but the PECs were maintained for 72 hours and then counted by a cell counter with trypan blue dye exclusion.
To assess apoptosis resistance, the PECs were seeded at a density of 5 £ 10 4 cells per well. After 24 hours of starvation (1% serum), the cells were incubated with I1 or I2 (50 and 500 nM) for 24 hours. Floating cells were collected and combined with adherent cells harvested by trypsin/EDTA treatment and stained with annexin V-FITC (BD Biosciences, Pont de Claix, France), and then analyzed by flow cytometry using a MACSQuant (Miltenyi Biotec). At least, 20,000 cells in each sample were counted by FACS analysis; additionally, floating cells were collected and combined with adherent cells harvested by trypsin/EDTA treatment, stained with Hoechst, and then analyzed by flow cytometry using a MACSQuant (Miltenyi Biotec).
To assess apoptosis resistance, the PECs were seeded at a density of 5 £ 10 4 cells per well. After 24 hours of starvation (1% serum), the cells were incubated with I1 or I2 (50 and 500 nM) for 24 hours. Floating cells were collected and combined with adherent cells harvested by trypsin/EDTA treatment and stained with annexin V-FITC (BD Biosciences, Pont de Claix, France), and then analyzed by flow cytometry using a MACSQuant (Miltenyi Biotec). At least, 20,000 cells in each sample were counted by FACS analysis; additionally, floating cells were collected and combined with adherent cells harvested by trypsin/EDTA treatment, stained with Hoechst, and then analyzed by flow cytometry using a MACSQuant (Miltenyi Biotec).
5
Quantifying Necroptosis by Flow Cytometry
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Ratio of PI-positive cells represented the level of necroptosis as previously described. Flow cytometry to measure the ratio of propidium iodide (PI)-positive cells was conducted according to the manufacturer’s instruction with some modifications. After OGD recovery (OGDR) for 12 h, the ratio of PI-positive cells was quantified using an AnnexinV-FITC Apoptosis Detection Kit by Flow Cytometry (MACSQuantTM, Miltenyi Biotech, Bergisch-Gladbach, Germany).
6
Multiparameter Flow Cytometry Assay
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For surface markers, the cells were stained in PBS containing 1% BSA with indicated antibodies for 30 min on ice. For intracellular markers, the cells were first fixed with Fixation Buffer (420801; Biolegend, San Diego, CA, USA) at 4 °C for 30 min and then stained in Permeabilization Wash Buffer (421002; Biolegend, San Diego, CA, USA) with relevant antibodies at 4 °C for 30 min. Foxp3 staining was conducted according to the manufacturer’s instructions for the Mouse Foxp3 Buffer Set obtained from BD Bioscience (San Diego, CA, USA). The following antibodies were used for the studies: APC anti-mouse CD45 (103112), PE anti-mouse F4/80 (123110), PerCP/Cy5.5 anti-mouse F4/80 (123128), FITC anti-mouse CD11c (117306), APC anti-mouse CD206 (141708), APC anti-mouse/human CD45R/B220 (103211), PerCP/Cy5.5 anti-mouse Ly-6G/Ly-6C (108427), FITC anti-mouse CD4 (100406), PerCP anti-mouse CD8a (100732), AlexaFluor 647 anti-mouse/rat/human Foxp3 (320014), PE anti-mouse/human CD44 (103008), and APC anti-mouse CD62L (104412) from Biolegend (San Diego, CA, USA), and PE-Cy7 anti-mouse CD11b (552850) from BD Bioscience (San Diego, CA, USA). Flow cytometry data were acquired on MACSQuantTM (Miltenyi Biotec, Auburn, CA, USA) and analyzed by FlowJo software (v10.5.3).
7
Isolation and Characterization of Mouse Adipose-Derived SVFs
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As above mentioned, SVFs were isolated from mouse adipose tissues. Flow cytometry was conducted as described51 (link). The SVFs were then stained in cold PBS containing 0.5% BSA and 2 mM EDTA (pH 7.4) with PE anti-mouse F4/80 (123110), FITC anti-mouse CD11b (101206) and APC anti-mouse CD11c (117310) for 30 min at 4 °C. After fixed with Fixation Buffer (420801) at 4 °C for 30 min, the cells stained in Permeabilization Wash Buffer (421002) with PE/Cy7 anti-mouse CD206 (141720) at 4 °C for 30 min. All the antibodies and reagents were purchased from Biolegend, San Diego, CA, USA. The antibody dilution was 1/400. The cells were subjected to flow cytometry analysis using a MACSQuantTM (Miltenyi Biotec, Auburn, CA, USA). Data analysis was performed using the FlowJo software (Tree Star Inc, Ashland, USA) as instructed.
8
Quantifying Intracellular Adriamycin Levels
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According to the method of our previous articles [22] , a cumulative ADR measurement assay was performed. Brie y, the cells were exposed to 5 μM ADR for 2 h and then washed with PBS. Next, ow cytometry was used to measure ADR uorescence to determine the intracellular ADR level.
Flow cytometric analysis of apoptosis MCF-7/ADR and MCF-7 cell apoptosis after ADR treatment was evaluated using the Annexin-V APC/7AAD Iodide Detection Kit (BD Biosciences, USA) according to the manufacturer's instructions. Cells were then analysed by MACSQuant (Miltenyi Biotec, Germany).
Flow cytometric analysis of apoptosis MCF-7/ADR and MCF-7 cell apoptosis after ADR treatment was evaluated using the Annexin-V APC/7AAD Iodide Detection Kit (BD Biosciences, USA) according to the manufacturer's instructions. Cells were then analysed by MACSQuant (Miltenyi Biotec, Germany).
9
Characterization of Transgenic T-cells and DCs
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Surface expression of transgenic TCRs was detected using an antibody directed against the murine constant TCR β region (mTRBC; Biolegend) and flow cytometric analysis. Anti-CD8 and anti-CD137 antibodies (Biolegend) were further used for T-cell characterization. HLA-DR (MHC class II), CD80, CD83, and CD86 for phenotyping of mDCs as well as the HLA-A,-B,-C antibody for measuring surface MHC expression on K562-HLA cells were purchased from Biolegend. Before antibody staining, mDCs were incubated for 10 min with a human Fc blocking antibody. Sytox blue (Thermo Fisher Scientific) was used for live/dead staining of cells, and flow cytometry measurements were performed at a MACSQuant (Miltenyi). Fluorescence-activated cell sorting (FACS) was performed on an Aria II (BD).
10
PDGFRB Expressing HEK293T Cell Assay
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HEK293T cells stably expressing PDGFRB (HEK293T-PDGFRB) were obtained from Zealand Pharma, Denmark. The cells were cultured in DMEM high glucose supplemented with 10% FCS, 1% sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine, 1% penicillin/streptomycin and 300 µg/mL of hygromycin. HEK293T cells were purchased from ECACC/Sigma Aldrich and cultured in DMEM high glucose with 10% FCS, 1% L-glutamine and 1% penicillin/streptomycin. Cells grown in flasks were detached by incubation with trypsin, counted and resuspended in PBS/10% FCS. Each sample contained 200,000 cells. The cells were incubated with VHH for one hour at 37 °C. After washing the cells two times in PBS/2% FCS/5 mM EDTA, PI was added to the buffer in a 0.1 µg/mL concentration as a live/dead stain. Flowcytometry measurements were performed using the MacsQuant from Miltenyi Biotec; data analyses were carried out using FlowJo version 10.
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