Thymi, spleens and MLNs were mashed into single-cell suspensions in PBS containing 1 % FCS and splenocytes were depleted of red blood cells using RBC lysis buffer (Sigma). Cell suspensions were incubated with mouse BD Fc Block™. CD4 + T-cells were enriched by the use of the mouse CD4 T lymphocyte enrichment set -DM from BD Biosciences or purified using BD IMag™ anti-mouse CD4 particles-DM (BD Biosciences). DP thymocytes were enriched using BD IMag™ anti-mouse CD8a particles-DM (BD Biosciences). BM from tibias and femurs was obtained by flushing with PBS containing 1 % FCS and depleted of red blood cells using RBC lysis buffer (Sigma). Stimulation of IFNΥ and IL-17 producing cells was performed in RPMI media (RPMI-1640 media containing penicillin/streptomycin, 50 μM β-mercaptoethanol and 10 % FCS) with 50 ng/ml PMA and 1 μg/ml Ionomycin (Sigma) for 4 h in the presence of GolgiPlug (BD Biosciences). IELs were prepared as described previously 26 . For FACS analysis cells were stained with antibodies (Abs) from BD Pharmingen directed against CD4 (RM4-5), CD8a (53-6.
Rbc lysis buffer
Manufactured by Merck Group
Sourced in United States, Germany
RBC lysis buffer is a laboratory reagent used to disrupt red blood cells (RBCs) and release their contents, including hemoglobin and other cellular components. This buffer facilitates the separation and analysis of cellular components in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (13 mentions)
L glutamine, by Thermo Fisher Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Sodium pyruvate, by Lonza (8 mentions)
Gentamycin, by Merck Group (7 mentions)
Its 1 solution, by Merck Group (7 mentions)
Rpmi 1640 medium, by Merck Group (7 mentions)
Dnase 1, by Merck Group (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Collagenase d, by Roche (6 mentions)
Non essential amino acids (neaa), by Lonza (5 mentions)
Hepes, by Thermo Fisher Scientific (5 mentions)
Liberase, by Roche (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Facscalibur, by BD (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
280 protocols using rbc lysis buffer
1
Isolation and Characterization of Murine Immune Cells
2
Single-cell Isolation from Mouse Spleen
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To separate single-cell suspensions, the collected individual spleens were passed through a 40-μm cell strainer in the presence of complete Roswell Park Memorial Institute (cRPMI) (Gibco BRL, Grand Island, NY, USA) medium supplemented with 10 % fetal bovine serum (Gibco BRL) and 1 % antibiotic (penicillin/streptomycin, Gibco BRL) using a sterile 5-mL syringe, and the cRPMI medium was removed by centrifugation (1500 × g for 3 min at 4 °C). Following this, single-cell suspensions were washed with phosphate-buffered saline (PBS) and centrifuged at 1500 × g for 3 min at 4 °C. To remove red blood cells (RBCs), single cells of spleen were incubated with 500 μL of RBC lysis buffer (Sigma-Aldrich) for 5 min at 4 °C, and the same volume of cRPMI medium as RBC lysis buffer was added and centrifuged (1500 × g for 3 min at 4 °C). The collected single cells were resuspended in cRPMI medium and used to analyze immune cells.
3
Tumor and Immune Cell Isolation Protocol
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The excised tumor mass was soaked in media containing enzymes (Tumor dissociation kit; Miltenyi Biotec, Bergisch Gladbach, Germany) to facilitate tumor dissociation, according to the manufacturer’s recommendations. The dissected lymph nodes adjacent to the tumor were mechanically squished using a 70 µm nylon cell strainer (SPL Life sciences, Gyeonggi-do, Korea) to isolate single cells and washed under running buffer (0.5% bovine serum albumin in PBS). The spleen was homogenized on a 70 µm nylon cell strainer, and red blood cells were lysed with 1×RBC lysis buffer (Sigma-Aldrich, Missouri, USA), filtered through a 70 µm nylon cell strainer, and counted.
Single cells were stained using Fixable Viability Stain 780 (BD, New Jersey, USA), Fixable Viability Stain 620 (BD), or zombie NIR Fixable Viability (BioLegend, California, USA) for 15 min at room temperature (25°C–27°C) and incubated with anti-CD16/32 antibodies (Thermo Fischer Scientific, MA, USA) for 15 min at room temperature. Then, the cells were placed on ice and surface-labled with monoclonal antibodies (online supplemental table 1 ) for 30 min. All events were acquired and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, California, USA). All flow cytometric data analyses were performed using Kaluza Analysis software (Beckman Coulter) and FlowJo software (BD).
Single cells were stained using Fixable Viability Stain 780 (BD, New Jersey, USA), Fixable Viability Stain 620 (BD), or zombie NIR Fixable Viability (BioLegend, California, USA) for 15 min at room temperature (25°C–27°C) and incubated with anti-CD16/32 antibodies (Thermo Fischer Scientific, MA, USA) for 15 min at room temperature. Then, the cells were placed on ice and surface-labled with monoclonal antibodies (
4
Isolation and Immunophenotyping of Bone Marrow Cells
5
Isolation and Stimulation of BAL Cells
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Broncho-alveolar lavage (BAL) was performed at necropsy by introducing 500 ml PBS into the lungs to recover BAL cells from both lung lobes. The BAL fluid was filtered through 2-layer fine gauze sheets into clean 50 mL centrifuge tubes, and stored at room temperature (RT) until further processing. The recovered cell suspensions underwent a series of washing with HBSS and centrifugation. To remove red blood cell (RBC) contamination the cell suspension was incubated for 5 minutes at RT in RBC lysis buffer (Sigma-Aldrich). Finally, the cells were resuspended in 5 mL RPMI-1640 media supplemented with 10% foetal bovine serum and penicillin/streptomycin. Cells were enumerated and either used for flow cytometry (see below) or plated out on 48-well plates at a concentration of 1.2 x 10 5 cells/well and incubated overnight (37 °C, 5% CO 2 ). The next day, cells were stimulated with either excretory/secretory (E/S) products from either A. suum or Trichuris suis, or lipopolysaccharide (LPS; 500 ng/mL) for 24 hours. Following stimulation, the supernatant was collected and stored at -20 °C before further analysis by ELISA.
6
Isolation of Suppressor Antigen-Presenting Cells
7
PLP-Specific T-Cell Activation Assay
8
Generation of BM Chimeric Mice
9
Evaluating Vaccinia Virus-Specific T Cell Responses
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Mouse spleens were isolated 14 days after VV immunization, mashed through 70 μm BD Falcon cell strainers and flushed with Roswell Park Memorial Institute (RPMI-1640) medium (Sigma Aldrich) containing 10% fetal calf serum (FCS), 1% streptomycin/penicillin, 1% sodium pyruvate, 1% non-essential amino acids (Gibco), and 0.1% β-mercaptoethanol. This medium was called complete T cell medium (CTCM). Splenocytes were lysed in red blood cell (RBC) lysis buffer (Sigma-Aldrich) and re-suspended in CTCM. Suspended cells were co-incubated for 72 h with previously reported VV immunogenic peptides (K3L, B2R, B8R, A8R, and A3L)25 (link) separately, and mesothelin peptide was used as control. IFN-γ production was assessed by ELISA (eBioscience) according to manufacturer’s protocol. The sequences of immunogenic peptides used in this experiment are K3L: YSLPNAGDVI; B2R: YSQVNKRYI; B8R: TSYKFESV; A8R: ITYRFYLI; and A3L: KSYNYMLL. All these peptides were synthesized by GL Biochem (Shanghai, China).
10
Splenocyte Stimulation Assay
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BDC2.5 animals were sacrificed, and spleens harvested and homogenized into single cell suspensions as described (18 (link)). Red blood cells were lysed using RBC lysis buffer (Sigma Aldrich). For in vitro experiments, 2.5 x 106 splenocytes were plated per well in 24 well plates and stimulated with 0.05 µM of their peptide mimotope ± 5 µM PFK15 soluble drug (Selleck), 0.2-1 mM 2-DG (Sigma Aldrich), 25-50 µM YN1 (Millipore Sigma), or 2.5-5 µM PFK158 (Selleck) for 24-72 hours. Cells and culture supernatants were collected for downstream flow cytometry, western blotting, ELISA, and lactate measurement analyses.
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