Bone marrow cells from wild-type C57BL/6J mice (Jackson Laboratory; stock number 000664) and Zbtb46-DTR mice were isolated by flushing femurs and tibias. Cells were passed through a 40μm cell strainer and subjected to red blood cell lysis (Sigma-Aldrich). After two washing steps, 1×106 per ml cells were seeded in 6-well plates in complete medium, consisting of RPMI 1640 medium supplemented with GlutaMAX (Life Technologies), 5% heat-inactivated FBS (Sigma-Aldrich), 1% penicillin/streptomycin (Life Technologies), 20U/ml polymyxin B (Fagron), 1mM sodium pyruvate (Life Technologies), 50μM β-mercaptoethanol (Life Technologies), and 10ng/ml GM-CSF (PeproTech). Cells were cultured at 37°C in a 5% CO2-humidified atmosphere for 6 days. At days 3 and 6, half of the culture medium was replenished. To study the effect of DT on immature BMDC, half of the cultured BMDC did not receive any maturation stimuli, and DT (2000ng/ml) was administered on day 6. On day 7, the remaining BMDC were activated by the addition of 1μg/ml lipopolysaccharide (LPS; Sigma-Aldrich) and 1000U/ml interferon-γ (IFN-γ; PeproTech), together with the addition of DT (2000ng/ml). Controls were not treated with DT. Twenty-four h after DT administration, cells were harvested for flow cytometric analysis.
Red blood cell lysis
Manufactured by Merck Group
Sourced in United States
Red blood cell lysis is a laboratory technique used to disrupt the cellular membranes of red blood cells, thereby releasing their contents. This process is commonly employed in various analytical and research applications to obtain samples for further analysis, such as the isolation of cellular components or the quantification of specific analytes.
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Lab products found in correlation
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Roche (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
96 well plate, by Corning (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primer express software v3, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Quantifast sybr green pcr master mix, by Qiagen (1 mentions)
Pan t cell kit, by Miltenyi Biotec (1 mentions)
Topcount, by PerkinElmer (1 mentions)
Recombinant granulocyte macrophage colony stimulating factor, by Thermo Fisher Scientific (1 mentions)
Cd90.2 microbeads, by Miltenyi Biotec (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
14 protocols using red blood cell lysis
1
Targeted Depletion of Dendritic Cells
2
Isolation and Culture of Mouse Splenocytes
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Splenocytes were prepared by aseptically removing the spleens from WT mice. Spleen tissues were homogenized into single-cell suspensions and subjected to red blood cell lysis (Sigma-Aldrich) according to instructions. Single splenocytes were seeded at a density of 1×106 cells in 96-well plate (Corning, New York, NY, USA) in culture containing RPMI 1640 media, 10% heat inactivated fetal bovine serum (FBS; Hyclone, South Logan, UT, USA), 1% penicillin/streptomycin, 10-mM L-glutamine, 10-mM Hepes, and 50-µM β-Mercaptoethanol.
3
Transcriptional Analysis of Antioxidant Genes
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Blood samples collected at 0, 120, and 360 min, were layered over Ficoll-paque Plus (GE Healthcare, Buckinghamshire, UK) and centrifuged. Following red blood cell lysis (Sigma-Aldrich, St. Louis, MO, USA), total RNA from MNC was isolated using RNeasy Mini Kit (QIAGEN, Netherlands). For reverse transcription of total RNA, high capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Waltham, MA, USA) was used. ViiA 7 Real-Time PCR System (Applied Biosystems) was used to perform gene expression assay. The PCR mix included 2 μL (10 ng) cDNA, 5 μL QuantiFast SYBR Green PCR Master mix (QIAGEN, Netherlands), and 0.1 μL of 100 μmol/L gene-specific primers (AIT Biotech, Singapore). Primers were designed using Primer Express software v3.0.1 (Applied Biosystems). All values were normalized to the expression of a housekeeping gene (GAPDH), which did not differ among the different phenotypes, time points and types of test meal. The panel of genes studied included, Nuclear factor, erythroid 2-like 2 (NRF2), Glutathione peroxidase (GPX3), Thioredoxin (TXN), Thioredoxin reductase 1 (TXNRD1), Superoxide dismutase (SOD- 1 and -2), Human neutrophil cytochrome –A light chain and –B light chain (CYBA and CYBB), Neutrophil cytosolic factor (NCF-1,-2, and -4), and Spi-1 (PU.1). Three sets of samples (2 lean subjects, 1 obese subject) were excluded from analysis due to poor quality of RNA.
4
Magnetically Isolated T Cell Proliferation
5
Splenocyte Activation and Modulation
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Splenocytes were prepared by aseptically removing the spleens from naive C57BL/6 mice. Spleens were homogenized into single-cell suspensions using a Stomacher 80 Biomaster blender (Seward, Davie, FL). The resulting suspension was centrifuged at 1600×g for 30 min and then subjected to red blood cell lysis (Sigma-Aldrich) according to the manufacturer’s instructions. The single splenocytes were collected and plated in a 96-well plate in RPMI 1640 media supplemented with heat inactivated 10% fetal bovine serum, 10 mM l -glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol, and 100 µg/ml penicillin/streptomycin at a density of 1 × 106 cells/well at 37 °C. Splenocytes were stimulated with PBS or 1 µg/ml SEB, followed by treatment with BBR (1, 2, 4, or 8 μM) or 100 nM tichostatin A (TSA) for 24 h. To assess activation, splenocytes collected from in vitro culture were stained with anti-mouse CD69 antibody (Biolegend, San Diego, CA, USA). Flow cytometry analysis was conducted in splenocytes using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the number of splenocytes was analyzed by Cell Quest Pro (BD Biosciences).
6
Isolation and Characterization of Immune Cells
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Splenocytes were filtered through 70 μm cell strainers and centrifuged at 300 g for 6 min at 4°C. Myeloid cells from liver were prepared as described previously [19 (link)]. Following red blood cell lysis (Sigma), cells were immunolabeled with fluorochrome-conjugated antibodies (S1 Table ) (Biolegend and BD) for flow-cytometry analysis (LSR II Sorb, Becton Dickinson). Intranuclear staining of ILC1 was performed using the Transcription Factor Buffer Set (BD Pharmingen). Blood was directly immunolabeled with fluorochrome-conjugated antibodies (S1 Table ) (Biolegend and BD) for flow-cytometry analysis (LSR II Sorb, Becton Dickinson) and subsequently treated with red blood cell lysing solution (BD). The gating of different cell populations is indicated in the supporting information section (S2 Fig ). Cell counts were determined using AccuCheck counting beads (Life technology).
7
Isolating Immune Cells from Murine Tissues
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When indicated, the spleen, liver, lung, and tumor were collectedfrom mice and incubated with 0.1 mg collagenase/DNase (Roche) for 30 min at 37 °C. Single-cell suspensions were prepared by mechanically disrupting the organs through a 70 µm cell strainer (Falcon). Tumor-infiltrating mononuclear cells, liver cells, and lung cells were isolated by centrifugation with 44% Percoll (GE Healthcare). Cells were then subjected to red blood cell lysis (Sigma-Aldrich). Blood was collected in PBS containing 2% FCS, 0.1% sodium azide, and 2.5 U/ml heparin. Peripheral blood mononuclear cells (PBMCs) were prepared by lysing erythrocytes with red blood cell lysis buffer. Mononuclear cells from the abovementioned organs were washed, resuspended in RPMI + 2% FCS (Gibco), counted and kept on ice until further analysis.
8
Isolation of Tumor-Infiltrating Immune Cells
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Mouse tumor tissue samples were harvested from in vivo experiments were processed for single-cell isolation. Tumors were cut into small pieces in ice-cold perfusion buffer (25 mM HEPES pH7.4, 0.5 mM EDTA in PBS) and transferred to gentleMACS C tube (Miltenyi Biotec GmbH) with four rounds of dissociation in digestion buffer with 0.5 mg/ml Collagenase IV (Invitrogen) and 0.1 mg/ml DNaseI (Roche) using gentleMACS dissociator (Miltenyi) according to instructions provided by the manufacturer. The cell suspensions were passed through 70 µm cell strainers prior to loading to percoll gradient mix to create the gradient. After centrifugation, the pellets were subjected to red blood cell lysis (Sigma Aldrich) and resuspended in FACS buffer (2% FBS PBS). Isolated cells were stained with anti-CD45-APC antibody (#55984, BD Biosciences) for 45 minutes on ice. LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Scientific) was used for exclusion of dead cells. Isolated cells were analyzed and the live CD45+ subpopulation was sorted on a BD FACSAriaIII cell sorter (BD Biosciences).
9
Isolation of Single-Cell Suspensions from Murine Tissues
10
Quantifying Immune Cell Subsets
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Spleens, tumors, and lymph nodes were gently dissociated under 40‐μm mesh cell strainer (BD Biosciences) for single‐cell isolation. After red blood cell lysis (Sigma‐Aldrich), single cells were washed and resuspended in PBS. These cells were labeled with fluorescence‐conjugated antibodies against mouse Gr1‐APC and CD11b‐PE (BD, PharMingen) to identify MDSC, or with PE‐conjugated CD4 and FITC‐conjugated FOXP3 to identify Treg cells and isotype‐matched controls. For intracellular staining, cells were fixed and permeabilized with Foxp3 staining buffer before incubation with the conjugated antibody or isotype control. Samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson) and data were analyzed using Flowjo software (version 7).
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