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6 protocols using ack rbc lysis buffer

1

Immunophenotyping of Blood Leukocytes

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Whole human blood was incubated in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 min at 37 °C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 min at room temperature, followed by BV421 CD45 (1:200, clone 2D1, #368522, Biolegend), APC CD14 (1:200, clone 63D3, #367118, Biolegend), BV605 CD16b (1:200, clone CLB-gran 11.5, #735143 BD), PE/Cy7 CD19 (1:200, clone HIB19, #302216, Biolegend), PE/Cy7 CD3 (1:200, clone HIT3a, #300316, Biolegend) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4 °C in the dark for 20 min, and washed in PBS/0.5% BSA. Neutrophils, lymphocytes, and monocytes were identified from peripheral blood cells of normal healthy donors with the following high dimensional immunophenotyping; Neutrophils (CD45+, CD3/CD19−, CD16b+, CD14), lymphocytes (CD45+, CD3/CD19+, CD16b−, CD14) and monocytes (CD45+, CD3/CD19−, CD16b−, CD14). Analysis was performed using an LSRII (BD) flow cytometer and FlowJo. v10 (Treestar).
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2

Lung Tissue Dissociation Protocol

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Lung lobes/large chunks from a single mouse were placed into a 2 mL microfuge tube and chopped into small (<2 mm) chunks with sterile scissors. 1 mL of digestion solution (TM Liberase at 4 units/mL (Roche, Basel, Switzerland) and DNAse Type I at 800 units/mL (ThermoFisher) made in sterile PBS) was added to each tube. Tubes were placed on a rotisserie at 37C for 20 min. Digest was then mechanically ground through 100um nylon filters (ThermoFisher) and placed in ACK RBC Lysis buffer (ThermoFisher) for 3 min at RT or as needed. Cells were pelleted and, if further cleanup is needed, a density gradient was used (debris removal solution, Miltenyi, Bergisch Gladbach, GER) according to manufacturer direction.
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3

Isolation and Immunophenotyping of Splenocytes

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Splenocytes were isolated from spleens of bone marrow chimeric mice by homogenization on 70 μm mesh cell strainers on tissue culture plates, followed by washing in RPMI media and incubating in ACK RBC lysis buffer (Thermo Fisher Scientific) for 4 minutes at 37°C to remove red blood cells. Cells were incubated with NIR Zombie (1:200, Biolegend) in PBS for live/dead staining for 15 minutes at room temperature, followed by PE CD45.1 (1:200, clone A20, eBioscience), APC CD45.2 (1:200, clone 104, Biolegend), BV 605 CD3e (1:200, clone 145–2C11, Biolegend), BV 650 CD19 (1:200, clone 6D5, Biolegend), FITC CD11b (1:100, clone M1/70, eBioscience), Percp Cy5.5 Ly6G/Gr-1 (1:200, clone RB6–8C5, eBioscience), eFluor 450 F4/80 (1:200, clone BM8, eBioscience) and Fc blocker (1:100, anti-mouse CD16/32,BD) at 4°C in the dark for 20 minutes, and washed in PBS/0.5% BSA. Analysis was performed using an LSRII (Becton Dickinson) flow cytometer and FlowJo. v10 (Treestar).
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4

Medulloblastoma Tumor Tissue Dissociation

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A medulloblastoma tissue sample was obtained after appropriate patient consent at the Lucille Packard Children’s Hospital (Stanford, CA), in accordance with Institutional Review Board protocols (Protocol ID #: 12625; IRB #: 4593 (Panel: 5)). Pathology of the tumor was confirmed upon histopathologic analysis by institutional neuropathologists prior to further analysis.
The tumor sample was mechanically dissociated in a solution containing Hank’s Balanced Salt Solution (HBSS), nonessential amino acids, sodium pyruvate, sodium bicarbonate, HEPES, Glutamax-1, antibiotic-antimycotic, DNase, and collagenase IV. All media components were from Cellgro, except for DNase and collagenase IV, which were from Worthington. The suspension was washed two times with HBSS, filtered through a 70-μm strainer and resuspended in a 0.9 M sucrose solution in HBSS without Ca2+/Mg2+ (Cellgro) to remove debris and dead cells. The cells were treated with ACK/RBC lysis buffer (Gibco), then washed twice in phosphate buffered saline (PBS). Cells were plated in tumor stem media (TSM) consisting of Neurobasal-A medium (Invitrogen), B27-A (Invitrogen), human bFGF (20 ng/ml) (Shenandoah Biotech), human EGF (20 ng/ml) (Shenandoah Biotech), human recombinant LIF (20 ng/ml) (Millipore), and heparin (10 ng/ml).
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5

Medulloblastoma Tumor Tissue Dissociation

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A medulloblastoma tissue sample was obtained after appropriate patient consent at the Lucille Packard Children’s Hospital (Stanford, CA), in accordance with Institutional Review Board protocols (Protocol ID #: 12625; IRB #: 4593 (Panel: 5)). Pathology of the tumor was confirmed upon histopathologic analysis by institutional neuropathologists prior to further analysis.
The tumor sample was mechanically dissociated in a solution containing Hank’s Balanced Salt Solution (HBSS), nonessential amino acids, sodium pyruvate, sodium bicarbonate, HEPES, Glutamax-1, antibiotic-antimycotic, DNase, and collagenase IV. All media components were from Cellgro, except for DNase and collagenase IV, which were from Worthington. The suspension was washed two times with HBSS, filtered through a 70-μm strainer and resuspended in a 0.9 M sucrose solution in HBSS without Ca2+/Mg2+ (Cellgro) to remove debris and dead cells. The cells were treated with ACK/RBC lysis buffer (Gibco), then washed twice in phosphate buffered saline (PBS). Cells were plated in tumor stem media (TSM) consisting of Neurobasal-A medium (Invitrogen), B27-A (Invitrogen), human bFGF (20 ng/ml) (Shenandoah Biotech), human EGF (20 ng/ml) (Shenandoah Biotech), human recombinant LIF (20 ng/ml) (Millipore), and heparin (10 ng/ml).
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6

Spleen Cell Isolation and ELISPOT Assay

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Mouse spleens were collected in 50 mL Falcon tubes decked with 70 μm nylon cell strainers and kept in ice. Spleens were gently passed through the cell strainer with the help of syringe plungers. Cells were then centrifuged at 700 g for 5 min at 4 °C. Cell pellets were resuspended with 1 ml Gibco™ ACK RBC Lysis buffer per spleen and incubated for 5 min at room temperature with occasional shaking. The reaction was then stopped by adding 30 ml of RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin and 50μM beta-mercaptoethanol. After centrifuging at 700 g for 5 min at 4 °C, the cell pellets were resuspended in 10 ml of ice-cold RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin and 50μM beta-mercaptoethanol, and plated at density of 250,000 cell/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Customized E6 or E7 peptide libraries (GenScript) were added to the wells at the final concentration of 5 μg/ml, unless otherwise indicated. After an overnight incubation at 37°C, plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions. All microplates were read under the CTL Immunospot Analyzer (ImmunoSpot®).
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