BMDMs were generated from the marrow of femurs and tibias from female C57BL/6 mice. Bones were rinsed in 70% ethanol and in PBS, and the bone marrow cells were flushed with PBS using a 25 G needle. After red blood cell lysis with RBC lysis buffer (Qiagen), cells were cultured in BMDM growth media, consisting of Iscove’s modified Dulbecco medium (IMDM, Sigma Aldrich), supplemented with 20% FBS, 1% P/S, and 20 ng/mL recombinant human M-CSF (R and D system), at a concentration of 1 × 106 cells/mL. The growth medium was refreshed on day 3. Differentiated macrophages were collected on day 7, and the purity determined to be more than 98% by multicolor flow cytometry, using cell surface markers CD11b and F4/80. The macrophages were cultured under different conditions for 24 h for specific macrophage polarization. For M1 polarization of macrophages, 100 ng/mL LPS (Invitrogen) and 40 ng/mL IFNγ (PeproTech) were used. For M2 polarizations, 40 ng/mL IL4 (PeproTech) was used. For adrenergic stimulation and blockade, (±) isoprenaline hydrochloride (isoprenaline) (Sigma Aldrich) or propranolol was dissolved in BMDM growth media and added to the cells.
Rbc lysis buffer
Manufactured by Qiagen
Sourced in Germany, United States
The RBC lysis buffer is a laboratory reagent designed to selectively lyse (break down) red blood cells (RBCs) in a sample. It is used to prepare cell suspensions for further analysis or processing by removing the RBCs, which can interfere with downstream applications. The buffer contains a combination of chemicals that disrupt the RBC membranes, allowing the cellular contents to be released and separated from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Gentlemacs dissociator, by Miltenyi Biotec (5 mentions)
Rbc lysis solution, by Qiagen (4 mentions)
Hanks balanced salt solution (hbss), by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Human ab serum, by Gemini Bio (2 mentions)
Dnase, by Roche (2 mentions)
Facsaria 2 and 3 machines, by BD (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
Brilliant buffer, by BD (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
70 m cell strainer, by BD (2 mentions)
70 μm cell strainer, by BD (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
33 protocols using rbc lysis buffer
1
Murine Bone Marrow-Derived Macrophage Polarization
2
Targeted Plasma DNA Sequencing
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Among 207 patients, target gene analysis was performed on 69 patients with sufficient blood at the time of examination for diagnosis. Whole blood samples were collected in Cell-Free DNA BCT tubes (Streck Inc., Omaha, NE, USA). After separation of plasma in the initial centrifugation, agranulocytes were separated by Ficoll gradient centrifugation and the granulocytes were separated from the bottom lymphocytes using RBC lysis buffer (Qiagen, Santa Clarita, CA, USA). Genomic DNA (gDNA) was isolated from granulocytes using a QIAamp DNA mini kit (Qiagen, Santa Clarita, CA, USA). Plasma DNA was obtained from 2 to 5 mL of plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen). The PBLs and plasma DNA libraries were created using a KAPA Hyper Prep Kit (Kapa Biosystems, Woburn, MA, USA) as described previously. In addition, capture baits for 66 genes selected from a 426 gene panel were customized and used for sequencing of cfDNA and their matched normal samples. After preprocessing, we identified somatic point mutations based on the previously reported iDES-enhanced CAPP-Seq with a minor modification [18 (link)]. The filtering steps to identify the variants were summarized as previously described [19 (link)].
3
Isolation and Cryopreservation of PBMCs and Splenocytes
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Peripheral blood was obtained from HIV-infected and uninfected individuals at the Massachusetts General Hospital (MGH), Boston. The study was approved by the MGH Institutional Review Board and written informed consent was obtained from all study participants prior to enrollment in the study. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation. Frozen PBMCs were thawed and were cultured in RPMI 1640 medium (Invitrogen) supplemented with HEPES, penicillin, streptomycin and 10% human AB serum (Gemini Bioproducts) (R10 medium).
Non-identifiable excess spleen tissue was obtained at Massachusetts General Hospital under approval by the Partners Human Resource Committee. These surgeries were performed for medical or surgical indications not related to hematologic or autoimmune diseases, with the exception of two patients who underwent splenectomy for refractory idiopathic thrombocytopenic purpura (ITP), which is currently an uncommon indication for such an intervention. Spleens were dissected into small fragments and subsequently mechanically disrupted to single cell suspension using a 70µm cell strainer and a syringe plunger. Following washing in R10 medium containing 2mM EDTA, cells were strained once more and erythrocytes were lysed using RBC lysis buffer (Qiagen). Cells were immediately cryopreserved for subsequent use.
Non-identifiable excess spleen tissue was obtained at Massachusetts General Hospital under approval by the Partners Human Resource Committee. These surgeries were performed for medical or surgical indications not related to hematologic or autoimmune diseases, with the exception of two patients who underwent splenectomy for refractory idiopathic thrombocytopenic purpura (ITP), which is currently an uncommon indication for such an intervention. Spleens were dissected into small fragments and subsequently mechanically disrupted to single cell suspension using a 70µm cell strainer and a syringe plunger. Following washing in R10 medium containing 2mM EDTA, cells were strained once more and erythrocytes were lysed using RBC lysis buffer (Qiagen). Cells were immediately cryopreserved for subsequent use.
4
Multicolor Flow Cytometry Analysis of Murine T Cells
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The thymus and spleen were dissected and smashed through a 70-µm strainer (Thermo Fisher Scientific, Waltham, MA, USA). Mouse peripheral blood was obtained by a retro-orbital puncture. RBC lysis buffer (Qiagen) was used to lyse the red blood cells, and the cells were suspended in FACS buffer (PBS containing 0.1% BSA). Organoid-derived T cells were harvested by adding hyaluronidase (300 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) to each well and were placed at 37 °C with a 5% CO2 incubator for hydrogel degradation overnight. The distribution of T cell subsets in the thymus or organoid was studied with four-color staining performed on freshly isolated thymocytes using the following antibodies: CD3 FITC (553061), CD4 APC (553051), CD8 PE (553032), and TCRβ Percp cy5.5 (H57-597) (all from BD Pharmingen, San Diego, CA, USA). T lymphocytes in the spleen and peripheral blood samples were analyzed using the following antibodies: CD3 APC (553066), CD4 FITC (553650), CD8 BV650 (563234), CD44 PE (553134) (all from BD Pharmingen), and CD62L Percp cy5.5 (104432) (Biolegend). All flow cytometry data were collected on a flow cytometer (CytoFLEX Beckman, CA, USA) and analyzed using FlowJo 10.5 software.
5
In Vivo Bioluminescent Imaging of Immune Cells
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All animal procedures and imaging protocols are performed under DFCI institutional IACUC guidelines. Splenocytes were harvested from NCr nude mice (Charles River Laboratories, Wilmington MA) and were treated with RBC lysis buffer (Qiagen, Valencia CA). The cells were then washed with HBSS prior to a 24 h culture period in RPMI 1640 containing 10 % FCS and 5 mg/mL of lipopolysaccharide (LPS, from Samonella enterica, Sigma Aldrich). The cells were collected and then stained with HBSS containing 1 μM of DiR. To generate light signal, the DiR-stained splenocytes suspension (100 μL, 1x107) was transferred into 100 μL of HBSS containing 2 μL of CSPD alkaline phosphatase substrate (Applied Biosystems) on a black 96-well plate. Untreated splenocytes cells were used as negative controls. For in vivo imaging, NCr nude mice received i.p. injection of 10 mg/kg LPS (Sigma Aldrich) in normal saline. In some animals, dexamethasone sodium phosphate (10 mg/kg, American Regent Inc., Shirley NY) was given 1 h prior to LPS challenge. 24 h later, the animal received an i.p. injection of 400 μL solution containing 50 % CSPD, 50 % reaction diluent, and 0.25 mg/mL DiR, prior to imaging. Cell and animal luminescence were scanned from 520 to 800 nm in 40 nm steps.
6
Lung and Immune Cell Isolation
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The tracheas were cannulated and washed twice with 1 ml of PBS before isolating the lungs. The lungs, lymph nodes (mesenteric, inguinal, axillary and mediastinum) and spleens were processed in RPMI 1640 medium. PBS was injected into the right ventricle to flush the circulating blood cells. The lungs were chopped into small pieces and digested with tissue digestion solution (0.5 mg/ml collagenase IV plus 8 μg/ml DNase I in HBSS containing 5% FBS) for 20 min before passing the tissue through a 70-µm cell strainer (BD Biosciences). Single cells were selected from the Aqua-stained (live) and CD45+ cell populations. RBC lysis buffer (Qiagen) was used to lyse the red blood cells. The bronchoalveolar lavage (BAL) fluid was stained with the Diff-Quik stain kit (Solarbio) according to the manufacturer’s instructions.
7
Tracking Metastatic Prostate Cancer
8
Immunophenotyping of PBMCs and Lung Cells
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Blood samples were processed from frozen PBMCs purified using standard ficoll separation. Samples were thawed in DNase-containing (25 units/ml) R10 (Sigma) at 37°C. Cells were rinsed and rested at 37°C for a minimum of one hour before undergoing red blood cell lysis by 5–10ml RBC lysis solution (Qiagen) for 20 minutes at room temperature. Cells were then stained with the appropriate antibody panel described below.
Blood for plasma isolation was centrifuged at 200 rpm for 10 minutes. The plasma layer was removed, frozen down in 1ml aliquots and stored at −80°C until needed. Later these samples were thawed at room temperature and vortexed thoroughly before usage.
Lung samples were processed from fresh tissue immediately following surgery. Resected tissues were washed with cold HBSS (Sigma) and dissected into smaller pieces. Tissues were rinsed again and resuspended in 10ml R10, containing DNAse (1μl/ml) and Collagenase (4μl/ml), and disassociated in a Gentle MACS dissociator (Miltenyi Biotec). Cells were rested in a shaking incubator at 37°C for 30 minutes and then further processed in the Gentle MACS dissociator. After further resting (30 mins at 37°C) and washing steps, cells were strained through a 70μm cell strainer and washed one final time. Cells were lysed using 5–10ml RBC lysis buffer (Qiagen) and stained for flow cytometry analysis.
Blood for plasma isolation was centrifuged at 200 rpm for 10 minutes. The plasma layer was removed, frozen down in 1ml aliquots and stored at −80°C until needed. Later these samples were thawed at room temperature and vortexed thoroughly before usage.
Lung samples were processed from fresh tissue immediately following surgery. Resected tissues were washed with cold HBSS (Sigma) and dissected into smaller pieces. Tissues were rinsed again and resuspended in 10ml R10, containing DNAse (1μl/ml) and Collagenase (4μl/ml), and disassociated in a Gentle MACS dissociator (Miltenyi Biotec). Cells were rested in a shaking incubator at 37°C for 30 minutes and then further processed in the Gentle MACS dissociator. After further resting (30 mins at 37°C) and washing steps, cells were strained through a 70μm cell strainer and washed one final time. Cells were lysed using 5–10ml RBC lysis buffer (Qiagen) and stained for flow cytometry analysis.
9
Handling and Infection of Bone Marrow Cells
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The detailed procedure of handling freshly obtained bone marrows have been described previously15 (link). Briefly, the sample from the original biological container, sometimes a syringe or IV bag, was removed. Care was taken to ensure the sample remained sterile. After initial blood smears to ensure cell viability, cells were treated with RBC lysis buffer (QIAGEN #386516) for 10 minutes on a mild shaker, after ensuring full RBC lysis we centrifuged the cells at 300 g for 6 min. Cells were counted and distributed into tubes before they were infected with Vero-derived dengue virus serotype 2, 16681 strain, at MOI = 0.1 for 2 hours with mild mixing every 20 minutes. Then the cells were washed with serum free RPMI by centrifugation and resuspension three times before they were distributed into different tubes and cultured in 2 ml of 10% FBS RPMI (Gibco #11875–093). Virus was harvested at assigned times.
10
Genomic DNA Extraction from Whole Blood
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DNA was extracted from blood collected in EDTA tubes. 3–5 ml of whole blood was incubated for 10 min in RBC lysis buffer (Qiagen GmbH, Hilden, Germany) and then centrifuged for 2 min at 2000 rpm to obtain white blood cell pellet, which was resuspended in 180 µl of residual supernatant and 20 µl of RNAse A (Qiagen GmbH, Hilden, Germany). Purification was then performed using the QiAamp DNA Blood mini kit on a QiaCube extraction device following the standard protocol.
Quantification was obtained using the Qubit dsDNA HS Assay (Life Technologies, CA, United States) and gel electrophoresis. The purity of DNA was verified through an evaluation of the 260/280 and 260/230 absorbance ratios on a Multiskan Go device (Thermo Scientific, Waltham, MA, United States).
At least 4 µg of DNA was needed per sample to use for quality control before sequencing at the CNRGH platform and to potentially prepare a second library in the event of technical problems. If the quantity or quality of DNA from a sample was insufficient, a new sample was requested from the center.
Quantification was obtained using the Qubit dsDNA HS Assay (Life Technologies, CA, United States) and gel electrophoresis. The purity of DNA was verified through an evaluation of the 260/280 and 260/230 absorbance ratios on a Multiskan Go device (Thermo Scientific, Waltham, MA, United States).
At least 4 µg of DNA was needed per sample to use for quality control before sequencing at the CNRGH platform and to potentially prepare a second library in the event of technical problems. If the quantity or quality of DNA from a sample was insufficient, a new sample was requested from the center.
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