Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood or leukopaks (New York Blood Center). Following red blood cell lysis with ACK (ammonium-chloride-potassium) Lysing Buffer (Lonza), human T cells were isolated from PBMCs (StemCell Technologies) and subsequently activated with 100 IU/mL of IL-2 and Dynabeads Human T-Activator CD3/CD28 at a bead:cell ratio of 1:5 (Thermo Fisher Scientific). Forty-eight hours after initial expansion, T cells were spinoculated with viral supernatant collected from 293Glv9 packaging cells on RetroNectin-coated plates on 2 consecutive days (Takara Clontech). All experiments were normalized for CAR+ viable cells.
Lysing buffer
Manufactured by Lonza
Lysing Buffer is a solution used in molecular biology and biochemistry laboratories to disrupt the cellular membrane and release the cellular contents, including proteins, nucleic acids, and other biomolecules, for further analysis or purification. The buffer is designed to effectively lyse a variety of cell types while preserving the integrity of the target molecules.
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Lab products found in correlation
Retronectin coated plate, by Takara Bio (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Pbmcs, by STEMCELL (1 mentions)
Allophycocyanin apc conjugated anti mouse cd11b, by Thermo Fisher Scientific (1 mentions)
Pe cy7 conjugated anti mouse cd3, by BioLegend (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Facsdiva version 6, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti mouse igg pe, by Jackson ImmunoResearch (1 mentions)
Facscanto, by BD (1 mentions)
Apc anti mouse cd49b, by BioLegend (1 mentions)
40 μm nylon mesh cell strainer, by BD (1 mentions)
5 protocols using lysing buffer
1
Efficient T Cell Transduction Protocol
2
Hematopoietic Cell Analysis by Flow Cytometry
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For the analyses of hematopoietic cells, a hypotonic lysis was performed to remove erythrocytes. 50 μl of blood was lysed using 500 μl of ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza, Walkersville) 10 min at room temperature. After the erythrocytes lysis, two washes with PBS were performed prior the incubation with the antibodies for 30 min at 4°C. Cells were stained with the following fluorochrome-coupled antibodies: Allophycocyanin (APC)-conjugated anti-mouse CD11b (1:300; cn.17–0112 eBioscience, USA) to label myeloid cells, phycoerythrin (PE)-conjugated anti-mouse B220 (1:100; cn.12–0452, eBioscience, USA) for B lymphocytes and phycoerythrin/cyanine (PE/Cy7)-conjugated anti-mouse CD3, 1:100; cn.100320, BioLegend, USA) for T lymphocytes. Immunofluorescence of labeled cells was measured using a BD LSR II flow cytometer. Dead cells and debris were excluded by measurements of forward- versus side-scattered light and DAPI (4′,6-diamino-2-phenylindole) (Sigma) staining. Gates for the respective antibodies used were established with isotype controls and positive cell subset controls. Data analysis was carried out using FACSDiva version 6.2 software (BD biosciences).
3
Murine T Cell Isolation Protocol
4
In Vitro IgE Quantification in Mice
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Whole blood cells from naive or peanut-sensitized mice were incubated after lysing of the erythrocytes (Lysing buffer; Lonza, Walkersville, Md) with serum of naive or CuMVtt-Ara h 1-immunized mice (1:5) together with peanut extract (1 mg/mL) in RPMI 164 for 30 minutes at room temperature. After washing, cells were stained with anti-mouse IgE-FITC (BD Becton Dickinson, Allschwil, Switzerland), anti-mouse CD49b-APC (BioLegend), and anti-mouse IgG-PE (Jackson ImmunoResearch, Cambridgeshire, United Kingdom). Measurements were performed with FACS Canto (BD Biosciences) and analysis with FlowJo software (FlowJo LCC).
5
Bone Marrow Transplant Experiments in Mice
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BMT experiments were performed using the same protocol as reported previously. 5 (link) Male, 8-week-old age mice with genotypes of miR-33b -/-Apoe -/-and miR-33b +/+ Apoe -/-were used as bone marrow (BM) donors. BM recipients were 8-week-old female miR-33b -/-Apoe -/-mice and miR-33b +/+ Apoe -/-mice. All mice used for BMT had an Apoe -/-background. BM donors were euthanized by cervical dislocation, and BM cells were collected by flushing femurs and tibias with PBS supplemented with 3% FBS. The suspension was passed through 40 μm nylon mesh cell strainer (BD Biosciences). Red blood cells were lysed using ACK (ammoniumchloride-potassium) lysing buffer (Lonza). BM cells were then washed twice with PBS supplemented with 3% FBS. To induce BM aplasia, recipients were irradiated with twice of 6 Gy within an interval of 3 hours (Cs 137 ; Gammacell 40 Exactor) and injected intravenously with 5×10 6 BM cells 6 hours after irradiation. 23, (link)24 (link) After BMT, mice were fed normal chow diet for 4 weeks and then switched to a WTD for 10 weeks. At 22 weeks old, mice were euthanized and analyzed. Successful hematopoietic reconstitution after BMT was confirmed by PCR amplification of the whole-blood genome and tail genome at the time of euthanization.
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