For the evaluation of antigen cross-presentation, WT and P2X7KO BM-DCs were stimulated for 24 h with H-AC-OVA and H-ACs-OVA-P2X7, in a 2:1 ratio of ACs/BM-DCs. As a positive control, BM-DCs were incubated with 5 μM of OVA257-264 peptide (SIINFEKL, Sigma-Aldrich, St. Louis, MO, USA) for 180 min at 37° C in a 5% CO2 atmosphere. After the pulse period, BMDCs labeling was performed with the anti-mouse- CD11c FITC and anti-mouse OVA antibodies (SIINFEKL/H-2Kb, clone eBio25D1.16 APC) from, both from eBioscience, San Diego, CA, USA. Total viable cells were evaluated by PI + exclusion. SIINFEKL/MHC-I complex was analyzed in the BM-DCs surface using the Accuri C6 Flow Cytometer and the information was processed using CFlow Plus software (eBioscience, San Diego, CA, USA)
Accuric6 flow cytometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AccuriC6 Flow Cytometer is a compact benchtop instrument designed for automated cell analysis and sorting. It utilizes state-of-the-art flow cytometry technology to rapidly analyze and quantify cellular characteristics, such as size, granularity, and fluorescence intensity. The instrument is capable of measuring multiple parameters simultaneously on a wide range of sample types, including cells, microparticles, and bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 flow cytometer, by BD (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
96 well plate, by Corning (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Anti mouse cd11c fitc, by Thermo Fisher Scientific (1 mentions)
Siinfekl, by Merck Group (1 mentions)
Flowcytomixtm pro, by Thermo Fisher Scientific (1 mentions)
Cpg odn, by InvivoGen (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Iscove s modified dulbecco s medium, by Merck Group (1 mentions)
Recombinant il 2, by Thermo Fisher Scientific (1 mentions)
Recombinant il 21, by Thermo Fisher Scientific (1 mentions)
Recombinant cd40l, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Countbright plus absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Ml349, by Bio-Techne (1 mentions)
Dead cell apoptosis kit with annexin 5 alexa fluor 488 and propidium iodide, by Thermo Fisher Scientific (1 mentions)
12 protocols using accuric6 flow cytometer
1
Evaluating Antigen Cross-presentation in BM-DCs
2
Bead-based Immunoassay Optimization
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Cell culture supernatants were concentrated ~ 10 times on columns and subjected to bead-based immunoassays according to the manufacturer’s instructions (BD 558301, 558347, 558296, 558299 560232). Data Acquisition and analysis was performed using a BD Accuri C6 flow cytometer and the eBioscience FlowCytomixTM Pro software respectively.
3
Evaluating B Cell Activation and Proliferation
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For each culture condition 9 • 10 5 B cells were cultured in tissue culture grade six-well plates preseeded with or without 1.8 • 10 5 MSC in Iscove's modified Dulbecco's medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (BioSera), 1% penicillin-streptomycin (Sigma-Aldrich), 1% l-Glutamine (Sigma-Aldrich), and 0.01% 2-bmercapthoethanol (Life Sciences). For studies requiring B cell activation and proliferation, B cells were activated using a cytokine cocktail of 25 ng/mL recombinant interleukin-10 (IL-10) (Peprotech), 100 U/mL recombinant IL-2 (Peprotech), 100 ng/mL recombinant IL-21 (Peprotech), insulin-transferrin-selenium (1: 1,000) (Gibco), 250 ng/mL recombinant CD40L (Peprotech), and 2.5 mg/mL CpG-ODN (Invivogen).
For experiments investigating B cell activation, purified CD19 + B cells were stained with CD19 and CD69. For experiments examining the effect of MSC on B cell proliferation, purified CD19 + B cells were stained with 10 mM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen). After 5 days, B cell proliferation was determined using an Accuri C6 flow cytometer (BD biosciences). For experiments investigating B cell viability, dual staining with FITC-conjugated Annexin V and Propidium Iodide (PI) was performed using a commercially available kit (eBioscience) to visualize apoptotic B cell populations on an Accuri C6 flow cytometer.
For experiments investigating B cell activation, purified CD19 + B cells were stained with CD19 and CD69. For experiments examining the effect of MSC on B cell proliferation, purified CD19 + B cells were stained with 10 mM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen). After 5 days, B cell proliferation was determined using an Accuri C6 flow cytometer (BD biosciences). For experiments investigating B cell viability, dual staining with FITC-conjugated Annexin V and Propidium Iodide (PI) was performed using a commercially available kit (eBioscience) to visualize apoptotic B cell populations on an Accuri C6 flow cytometer.
4
Assessing Talazoparib Cytotoxicity in CLL Cells
5
Assessing Talazoparib Cytotoxicity in CLL Cells
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Peripheral blood mononuclear cells were isolated from blood samples collected from patients with a new or existing diagnosis of CLL, irrespective of the stage of disease or time/type of treatment from two Birmingham hospitals (Heartlands and Queen Elizabeth). This study was approved by the South Birmingham Ethics Committee (REC number 10/H1206/58), performed according to institutional guidelines and written consent was obtained from all participants.
Primary chronic lymphocytic leukemia (CLL) cells (5 x104) and CD40L-expressing mouse embryonic fibroblasts (5 x103) were seeded in each well of a 96-well plate (Corning) in 100 μl of RPMI media supplemented with 10% FBS (Sigma-Aldrich, UK) and 25 ng/ml IL-21 (eBioscience)47 (link). After 24 h, 200 μl more media was gently added and cells were incubated for another 48 h. The media was then aspirated, replaced with 200 μl of media containing talazoparib and cells were incubated for a further 72 h. The cytotoxic effect of PARPi was determined by propidium iodide exclusion as measured by flow cytometry with an Accuri C6 flow cytometer (Applied Biosystems). Only cells, which entered the cell cycle upon stimulation (as determined by forward- and side-scatter FACS profiles), were considered for analysis. Data was expressed as a surviving fraction relative to untreated cells, for gating strategies seeSupplementary Fig 2 .
Primary chronic lymphocytic leukemia (CLL) cells (5 x104) and CD40L-expressing mouse embryonic fibroblasts (5 x103) were seeded in each well of a 96-well plate (Corning) in 100 μl of RPMI media supplemented with 10% FBS (Sigma-Aldrich, UK) and 25 ng/ml IL-21 (eBioscience)47 (link). After 24 h, 200 μl more media was gently added and cells were incubated for another 48 h. The media was then aspirated, replaced with 200 μl of media containing talazoparib and cells were incubated for a further 72 h. The cytotoxic effect of PARPi was determined by propidium iodide exclusion as measured by flow cytometry with an Accuri C6 flow cytometer (Applied Biosystems). Only cells, which entered the cell cycle upon stimulation (as determined by forward- and side-scatter FACS profiles), were considered for analysis. Data was expressed as a surviving fraction relative to untreated cells, for gating strategies see
6
Tracking Donor T Cell Responses
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TCR-Tg cells (5 × 102–6 × 103) were injected i.v. into secondary naive B6 hosts immunized 24 hours later with B/c DST i.v. Within each experiment, the number of transferred cells was similar between mice. Cell concentration was confirmed by counting on either an Accuri C6 flow cytometer or an LSR 4 to 12 flow cytometer using CountBright Plus Absolute Counting Beads (Invitrogen) prior to injection. Five days after DST, 5 × 106 splenocytes were isolated from the secondary hosts, then stained with a viability dye and fluorophore-conjugated anti-CD4, anti-CD8, anti-CD45.1, and anti-CD45.2. The number of CD45.1+CD4+ cells was calculated per mouse.
7
Antiproliferative Effects of Palmostatin B, ML348, and ML349
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Palmostatin B was purchased from Merck Millipore (178501). ML348 and ML349 were purchased from Tocris Bioscience (USA). Cells were plated in 96-well plates with a density of 4000-8000 cells per well and incubated for 24 h at 37°C with 5% C02. Then cells were treated with increasing drug concentrations and their combinations. Cell viability was measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega; Wisconsin, USA) according to the manufacturer's protocol. Luminescence was measured on the SynergyHT plate reader (BioTek, Vermont, USA) using Gen5 software (Version 1.11.5). For apoptotic assays 0.1–0.2 × 106 cells were plated in 12-well plates and treated with DMSO or an inhibitor. After 72 hours apoptosis was assessed using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and Propidium Iodide according to the manufacturer's instructions (Invitrogen, V13241) with the AccuriC6 Flow Cytometer using the CFlow software (Ver. 1.0.227.4). Concentrations of drugs resulting in 50% decrease in cell viability relative to DMSO treated controls (GI50) were calculated with CalcuSyn software (Biosoft, Cambridge, UK, Version 2.1).
8
TRAIL-R1/R2 Surface Expression
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To detect surface expression of TRAIL-R1 and -R2, 2 × 105 cells were detached using 2 mM EDTA, washed in PBS, and resuspended in PBS (0.1% BSA) containing antibodies for TRAIL-R1/DR4, TRAIL-R2/DR5 (eBiosciences) or IgG control. AlexaFluor 647 (Invitrogen) secondary antibody was used to detect the levels of TRAIL-R1/2, and mean fluorescence intensity was measured via Accuri C6 Flow Cytometer.
9
Synergistic effects of trametinib and metformin
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Trametinib and metformin were purchased from Selleck Chemicals (Houston, Texas, USA). For cell viability assays, cells were plated in 96-well plates with a density of 4000-8000 cells per well and incubated for 24 h at 37 °C with 5% C02. Then cells were incubated with increasing drug concentrations and their combinations. Cell viability was measured with the CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Promega; Madison, Wisconsin, USA) according to the manufacturer's protocol. Luminescence was measured on the SynergyHT plate reader (BioTek, Vermont, USA) using Gen5 software (Version 1.11.5). For apoptotic assays, cells were plated in 12-well plates and treated with DMSO, Trametinib, metformin or combinations. After 72hrs apoptosis was assessed using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide according to the manufacturer's protocol (Invitrogen; V13241) with the AccuriC6 Flow Cytometer using the CFlow software (Version 1.0.227.4).
10
Apoptosis Assay of Drug Combinations
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Cells were plated in 12-well plates and after 24 hour incubation treated with DMSO, trametinib, GSK2126458 or their combinations. After 72hrs apoptosis analysis was performed using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide according to the manufacturer's protocol (Invitrogen; V13241) with the AccuriC6 Flow Cytometer using the CFlow software (Version 1.0.227.4).
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