All normal samples were processed in accordance with the scientific protocol accepted by the local ethical committee (N-20080062MCH). Thy and BM from sternum were surgically obtained pairwise from seven patients undergoing cardiac surgery (Supplementary Table E1, online only, available at www.exphem. org). Thy tissue was cut into pieces and homogenized in phosphate-buffered saline (PBS) using a syringe. The suspensions were washed once in PBS and enriched by gradient centrifugation on Ficoll-Paque Plus (GE Health Care, Uppsala, Sweden) for mononuclear cells (MNCs) in accordance with the manufacturer's instructions. The BM was homogenized in 2 mL of PBS using a syringe. The red blood cells were lysed by adding 20 mL of Easylyse (DAKO, Glostrup, Denmark), and they were incubated for 45 minutes at room temperature (RT). The samples were washed once in PBS before passing through a 40-mM filter to remove debris and aggregates. Peripheral blood mononuclear cells were isolated in a LeucoSep tube (Greiner Bio-One, Frickenhausen, Germany) after red blood cell lysis (Easylyse DAKO, Glostrup, Denmark) according to the manufacturer's instructions. Tonsils were obtained by routine tonsillectomy as described previously [30] .
Easylyse
Manufactured by Agilent Technologies
Sourced in Denmark, United Kingdom
Easylyse is a compact and versatile sample preparation system designed for automated DNA/RNA extraction and purification. It offers a streamlined workflow to efficiently process a wide range of sample types, including cells, tissues, and bodily fluids. The system is optimized for reliable and reproducible results, providing researchers with a user-friendly platform to support their analytical requirements.
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Lab products found in correlation
Leucosep tube, by Greiner (2 mentions)
7 aad solution, by Thermo Fisher Scientific (2 mentions)
96 well u bottom plate, by BD (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Ly6g pe cy7, by BD (1 mentions)
Nk1.1 apc, by BD (1 mentions)
Abx pentra 60c, by Horiba (1 mentions)
Cd11b bv510, by BD (1 mentions)
Turk s solution, by Merck Group (1 mentions)
Cd45 apc fire 750, by BD (1 mentions)
F4 80 pe, by BD (1 mentions)
Ly6c fitc, by BD (1 mentions)
Cd19 pe, by BD (1 mentions)
Cd8 pe cy7, by BD (1 mentions)
Cd4 bv421, by BD (1 mentions)
Cd11c bv421, by BD (1 mentions)
Summit v4, by Beckman Coulter (1 mentions)
Cyan adp, by Beckman Coulter (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Precision count beads, by BioLegend (1 mentions)
19 protocols using easylyse
1
Isolation of Immune Cells from Thymus and Bone Marrow
2
Flow Cytometric Analysis of CD14+ Monocytes
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Blood samples were lysed with EasyLyse (DakoCytomation, code-Nr.S2364, Glostrup, Denmark) and subsequently stained with fluorescein isothiocyanate-labeled CD14 monoclonal antibody (Invitrogen, MHCD1401, Carlsbad, CA, USA) in the wells of a flexible 96-well U-bottom plate (BD Biosciences, San Jose, CA, USA) at 37 °C for 20 min. The cells were washed with phosphate-buffered saline containing 1% fetal calf serum and then analyzed using a CyAn ADP analyzer (Beckman Coulter, Indianapolis, IN, USA). Results were expressed as the mean of fluorescence intensity. Four hundred and one subjects who did not have cancer and who were non-exposed (radiation dose <0.005 Gy) were randomly selected from the IMG cohort members with the aim of excluding the effects of cancer and radiation on the relationship between CD14 genotype and mCD14 levels. The mCD14 levels were considered as the dependent variables to be analyzed after taking the logarithm at the base 10. This logarithmic transformation was applied to ensure the normality of the error distribution.
3
Immunophenotyping of Mouse Blood Cells
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The heparinized blood from each animal was divided into two parts. A volume of 150 µl was designated for blood count and measured using an ABX Pentra 60 C+ hemoanalyzer (Horiba, Kyoto, Japan). The rest of the blood (200–300 µl) was lysed using EasyLyse™ (Dako, Glostrup, Denmark). The amount of cells in suspension was as determined by Turk’s solution (2% acetic acid; Sigma-Aldrich) using a hemocytometer chamber. Cells (5 × 105/100 µl) were marked with two panels of monoclonal antibodies. The first panel was delineated to detect T, B, and NK lymphocytes (CD3ϵ, CD4, CD8, CD19, and NK1.1). The second panel was designed for determination of monocytes and neutrophils (CD11b, F4/80, Ly6C, and Ly6G). The following monoclonal antibodies with fluorochromes for flow cytometry analysis were purchased from BioLegend (San Diego, CA, United States): anti-mouse CD3ϵ–FITC, CD4–BV421, CD19–PE, CD11c–BV421, CD45–APC/Fire™750, F4/80–PE, and from BD Bioscience: CD8–PECy7, CD11b–BV510, Ly6C–FITC, Ly6G–PECy7, and NK1.1–APC.
4
Apoptosis Assessment in Blood Cells
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In apoptosis assessment experiment, 300 μl of heparin (Zentiva, Czech Republic) were added to peripheral blood obtained from 10 healthy volunteers and radiation exposed patients. The peripheral blood was kept at the room temperature and lysed by the EasyLyse, erythocyte lysing reagent (DAKO, Glostrup, Denmark) according to manufacturer´s instructions. The cell suspension density was set to 5 x 106 cells/ml in diluted Binding buffer (1:10). Lymphocytes, lymphocytes and monocytes (PBMC), and granulocytes were stained with monoclonal antibody Annexin V-FITC (BD Biosciences Pharmingen, San Jose, CA, USA) for 10 min on ice. Five μl of propidium iodide (250 μg/ml) were added after one washing step in an ice cold Washing and staining buffer. Data were acquired on CyAn ADP flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analysis was performed using the Summit v4.3 software (Beckman Coulter), where lymphocytes, PBMC, and granulocytes were divided based on forward scatter / side scatter characteristics and cell death was assessed in lymphocytes, PBMC, and granulocytes populations. The protocol is available on-line at https://dx.doi.org/10.17504/protocols.io.mjhc4j6 .
5
Quantifying Immune Cell Populations
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The quantification of cellular populations was performed on whole blood by using the following antibodies: CD25 (3C7), Ly6G (1A8), CD19 (1D3), CD335 (NKp46), Ter119 (TER-119), CD41 (MWReg30), CD11b (M1/70), CD11c (HL3), Ly6C (AL-21), CD3e (145-2C11), and CD127 (SB/199) (Becton Dickinson). Erythrocytes were then lysed using Easylyse (Dako). The cells were labeled with Di Aminido Phenyl lndol (DAPI) (Sigma-Aldrich) to exclude dead cells, and precision count beads (BioLegend) were added after staining to calculate the absolute number of cell subpopulations. Gating strategies are displayed in SI Appendix, Fig. S2 . A Fortessa ×20 flow cytometer (Becton Dickinson) was used to run all samples. The data were analyzed using Kaluza 2.0 software.
6
Quantifying Mitochondrial Mass in T Cells
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Whole blood was first labeled with a CD3e (145-2C11) antibody, and the erythrocytes were then lysed using Easylyse (Dako). The quantification of mitochondrial mass into T cells was performed using the MitoTracker Green probe (Invitrogen). The cells were incubated for 20 min at 37 °C before washing in phosphate-buffered saline (PBS) and running in a Fortessa ×20 flow cytometer (Becton Dickinson).
7
Quantifying T-cell Proliferation in Mice
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The mice were injected with 20 mg 5-ethynyl-2′-deoxyuridine (EdU) (Invitrogen) 24 h before euthanasia. The spleens were harvested, perfused with Roswell Park Memorial Institute medium fetal bovine serum-10%, and filtered at 70 µm. The erythrocytes were lysed using Easylyse (Dako), and the cells were washed in PBS BSA-1%. A total of 0.5 × 106 cells were incubated with CD3e antibody (145-2C11, Becton Dickinson) for 20 min at 4 °C. The cells were washed in PBS bovine serum albumin-1% (BSA-1%) and fixed for 15 min at room temperature with Click-iT fixative (Click-iT Plus EdU Flow Cytometry Assay Kit, Life Technologies). The cells were washed in PBS BSA-1% and incubated with Click-iT saponin-based permeabilization and wash reagent and Click-iT Plus reaction mixture for 30 min at room temperature. The cells were washed with Click-iT saponin-based permeabilization and wash reagent. The cells were resuspended in Click-iT saponin-based permeabilization and wash reagent with Dapi at 10 µg/mL and incubated for 20 min at room temperature before running the sample in a Fortessa ×20 flow cytometer.
8
Isolation of Peripheral Blood Mononuclear Cells
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PBMNC (n = 6 ) were isolated by diluting the peripheral blood sample 1:1 in phosphate-buffered saline (PBS) and placing the sample in a LeucoSep tube (Greiner Bio-One, Frickenhausen, Germany), according to the manufacturer’s instructions. The mononuclear cells (MNCs) were washed once and the red blood cells were lysed by adding 9 ml of Easylyse (DAKO, Glostrup, Denmark) to the pellet and incubating the sample for 15 minutes at room temperature (RT). The MNCs were washed twice in PBS and sorted fresh.
9
T Cell Proliferation Assay with CFSE
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The splenocytes were isolated, mashing spleen with a plunger end of syringe into a 40-µm cell strainer. The erythrocytes were lysed using Easylyse (Dako). The cells were then labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE; Intercom). A 96-well round-bottom plate was precoated with an anti-CD3e (145-2C11, 5 µg/mL, eBioscience) for 2 h at 37 °C. The T cells (105 cells/well) were added and incubated for 72 h with an anti-CD28 antibody (37.51, 2.5 µg/mL, eBioscience) or complete media for control well in a humidified environment with 5% CO2 at 37 °C. After 3 d of culture, the cells were harvested and labeled with CD4 (GK1.5), CD8 (53-6.7) antibodies (BD Biosciences), and FVS780 (BD Biosciences) for cell viability. CFSE dilution was assessed by flow cytometry on a Fortessa X-20, and results were analyzed with ModFit LT software.
10
Whole Blood Stimulation and CD62L Expression
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Whole blood was stimulated as previously described,26 (link) with little modification in relation to the used agonists. For each stimulation, 100 μL heparinized blood was treated with Polymyxin B, 10 µg/mL for 20 minutes (except the sample stimulated with lipopolysaccharide) and hereafter, incubated at 37°C in a 5% CO2 atmosphere for 1 hour with the individual agonists Pam3csk48 (Invivogen, San Francisco, CA; TLR1/2), Pam2csk4 (Invivogen; TLR2/6), lipopolysaccharide (Invivogen; TLR4), flagellin-ST (Invivogen; TLR5), CL097 (Invivogen; TLR7/8), ssRNA40 (LyoVec, Invivogen; TLR8) and with polymyxin A (Sigma-Aldrich, St. Louis, MO) or tumor necrosis factors a (TNFa; R&DSystems, Minneapolis, MN) as positive controls. After activation, the erythrocytes were lysed in EasyLyse (Dako) and washed twice in phosphate-buffered saline. The cells were incubated with anti-human CD62L or isotype control (BD Bioscience Pharmingen 555544 resp. 555749) for 20 minutes in 4°C.26 (link)
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