All the materials used in this research were procured from different sources. Beging Mesochem Ltd. (Beiging, China) provided the apremilast, while Sigma Aldrich provided the LPS to induce-lung injury, Broadford reagent for protein measurement, and hematoxylin and eosin (H&E) for histological staining (St Louis, USA). EMD Millipore (Massachusetts, USA) provided ELISA kits for the biochemical assay. RBC Lysis Buffer (10X) and antibody for flow were obtained from BioLegend® Inc (SanDiego, CA). The mRNA gene expression primer, PCR Master Mix, High-capacity cDNA reverse transcription kits, and SYBR® Green were procured from Applied Biosystems (Paisley, UK). Life Technologies (Grand Island, USA) provided the TRIzol®. Santa Cruz (Dallas, USA) provided the primary and secondary antibodies used in the western blot study. PVDF blot transfer membrane (Immobilon®-FL) purchased from Merck Millipore Ltd. (Oakville, Canada) and Chemiluminescent HRP Substrate for Western blot identification kits was purchased from Millipore Corporation (Billerica, USA). All of the other chemicals used were analytical grade and came from commercial sources.
Rbc lysis buffer 10x
Manufactured by BioLegend
Sourced in United States
RBC Lysis Buffer (10X) is a concentrated solution designed to lyse red blood cells (RBCs) in a variety of biological samples. The buffer is formulated to effectively remove RBCs, which can interfere with downstream applications, such as flow cytometry and immunoassays. When diluted to 1X working concentration, the buffer is suitable for use in various cell preparation and analysis protocols.
Automatically generated - may contain errors
Lab products found in correlation
Pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Falcon tube, by BD (1 mentions)
Collagenase d solution, by Roche (1 mentions)
Heparin, by B. Braun (1 mentions)
Lsrfortessa 2 cell analyzer, by BD (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Intracellular staining permeabilization wash buffer 10x, by BioLegend (1 mentions)
Hla dr, by BioLegend (1 mentions)
Cxcr4, by BioLegend (1 mentions)
Sybr green and cdna kits, by Thermo Fisher Scientific (1 mentions)
Cxcr7, by BioLegend (1 mentions)
Gata 3, by BioLegend (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
8 protocols using rbc lysis buffer 10x
1
Apremilast Attenuates LPS-Induced Lung Injury
2
Isolation of Lymphocytes from NOD Mice
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NOD.c3c4 mice (n = 3–5) and NOD mice (n = 3–5) were killed at the age of 10 weeks. Thymus and spleen were harvested and put in cold phosphate‐buffered saline (PBS). The liver was perfused with 4 to 10 mL cold PBS, harvested, passed through a 70‐μm nylon mesh, and washed two times (Zeissig et al. 2013 ). The cell suspension was overlaid on a 40%/60% Percoll® (Sigma‐Aldrich, St.Louis, MO) density gradient and centrifuged at 700g for 20 min at 4°C (without brakes). The lymphocyte layer was collected and washed. The spleen was pressed through a 40‐μm mesh, the cells were washed and the red blood cells were lysed with RBC Lysis Buffer (10X) (BioLegend, San Diego, CA). The thymus was pressed through a 40‐μm mesh and the cells were washed and collected.
3
Isolation of Mouse Splenocytes
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After euthanasia, mouse spleens were harvested. The spleens were washed with PBS and mashed through a 40 μm cell strainer using a sterile syringe plunger into a 60 mm petri dish. The single cell suspension was washed with complete RPMI-1640 media (containing 10% fetal bovine serum and 1% penicillin-streptomycin), collected into a Falcon tube, and treated two times with red blood cell lysis buffer (RBC lysis buffer (10X); BioLegend; #420301), according to manufacturer’s protocol. The cell pellet was resuspended in 2 mL of complete RPMI media, and the cell number was counted. Splenocytes were then applied for flow cytometry or ELISpot assays.
4
Multimodal Analysis of Tumor Cells
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Peripheral blood (PB), BM, lung, liver, gut and spleen samples were isolated and analyzed for persistence of human tumor or effector cells: BM was flushed out of the femur and tibia of mice. PB and BM were incubated with red blood cell lysis buffer (RBC Lysis Buffer (10x), BioLegend) according to the manufacturer´s instructions and washed once with PBS. The organs were cut in representative halves, and one half was preserved in formaldehyde for fluorescence microscopy. The other half was incubated with collagenase D solution (Roche, Basel, Switzerland), filtered through a 70 µm cell strainer, and washed with PBS. Aliquots of the cell suspensions were analyzed by flow cytometry and quantitative polymerase chain reaction (qPCR). Sample identity was blinded for the executors of the consecutively analyses.
5
Quantifying Leukemia Progression via Circulating Tumor Cells
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Leukemia progression was evaluated by weekly quantification of peripheral blood circulating tumour cells. Blood (70–100 µL) was collected from facial vein and stored in tubes containing 10 µL of Heparin (500un/mL, B.Braun, Germany). In each blood sample, a defined amount of beads (Coulter CC Size Standard L10) was added for absolute cell quantification and Red Cells Lysis Buffer 1x(RBC Lysis Buffer 10x, Biolegend, USA) was added in multiple rounds until complete erythrocyte clearance. Cells were stained with LIVE/DEADTM dead cell staining kit (InvitrogenTM, NY, USA) for 15 min at 4 °C. Total GFP + Live/Dead– cells were quantified by Flow cytometry on an LSR FortessaII Cell Analyzer (BD Biosciences). Data was analyzed with FlowJo X 10.0.7 software (TreeStar, USA) and the results shown as the absolute numbers per mL of blood. Animals were assigned randomly to treatment groups upon reaching a minimum level of 100 GFP+ CD45+ viable cells/mL of blood (treatment threshold).
6
Comprehensive Immune Cell Analysis
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Fluoroisothiocyanate, PE/dazzle, allophycocyanin, and phycoerythrin-labeled CD3, CD4, CD8, CXCR4, CXCR7, CD45R, HLA-DR, GATA3, Ki-67, Helios, and FOXP3 human monoclonal antibodies, RBC lysis buffer (10X), intracellular staining permeabilization wash buffer (10X), and fixation buffer were purchased from BioLegend (San Diego, USA). GolgiStop was purchased from BD Biosciences (San Diego, USA). RPMI 1640 medium, phorbol myristate acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was purchased from Life Technologies (Paisley, UK). SYBR green and cDNA kits were purchased from Applied Biosystems (Foster City, CA, USA). Primers were synthesized from GenScript (Piscataway, NJ, USA).
7
Bone Marrow Cell Isolation Protocol
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After euthanasia, femurs were collected and washed with PBS several times. Muscle fibers and connective tissues were removed with forceps. The marrow was flushed from the bone using a syringe and collected into a dish with PBS. The marrow clot was pipetted and filtered through a 40 μm cell strainer and collected into a Falcon tube. The single cell suspension was pelleted at 300 × g for five minutes. Supernatant was discarded, the pellet was resuspended, and the cells were treated with red blood cell lysis buffer (RBC lysis buffer (10X); BioLegend; #420301), according to the manufacturer’s protocol. The suspension was pelleted at 300 × g for five minutes and resuspended in culture media. B cells were analyzed via flow (Cytoflex LX).
8
Isolation and Analysis of Murine Myeloid-Derived Suppressor Cells
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Antibodies used are listed in the Suppl. Table 1. FoxP3 staining Buffer Set was purchased from eBioscience. Collagenases III and IV and Trypan Blue 0.5 % were obtained from Biochrom AG. DNAse I was purchased from Roche. Dulbecco’s PBS Solution (10x), HBSS Buffer, and RPMI 1640 were obtained from PAA Laboratories GmbH. RBC Lysis Buffer (10x) was purchased from BioLegend and Hyaluronidase by Linaris GmbH. CD11b MicroBeads as well as Myeloid-Derived Suppressor Cell Isolation mouse kit were purchased by Miltenyi Biotec GmbH. Milliplex® MAP Kit, Mouse Cytokine/Chemokine Magnetic Bead Panel, was purchased from EMD Millipore Corporation Merck KGaA. FluoSpheres®Carboxylate, Yellow-green (505/515) conjugated, was obtained from Life Technologies. CFSE (5-(and6)-Carboxyl-fluorescein diacetate succinimidyl eyter, CFDA SE) and CD274 (B7-H1) Functional Grade Purified antibody were purchased from eBioscience.
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