Bone marrow was isolated from the tibia and femur of one 8 week old C57BL/6 mouse and erythrocytes were removed with Red Blood Lysing Buffer (Sigma-Aldrich). Then, 6 × 106 isolated bone marrow cells were cultured in a 6 well dish in RPMI medium supplemented with 10% heat inactivated FCS, 5 mM HEPES (Invitrogen), 50 μM β-Mercaptoethanol (Sigma), 100 U/ml of Penicillin and Streptomycin (Invitrogen) and GM-CSF (10 ng/ml) (Peprotech) for 5 days at which point BMDCs were harvested and processed for T cell signaling.
Red blood lysing buffer
Manufactured by Merck Group
Sourced in United States
Red Blood Lysing Buffer is a laboratory reagent used to selectively lyse (break open) red blood cells in a sample, allowing for the isolation and analysis of other cell types, such as white blood cells. It is a key component in various hematology and immunology procedures.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by Thermo Fisher Scientific (2 mentions)
Facscablibur system, by BD (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Ficoll histopaque 1077, by Merck Group (1 mentions)
5 protocols using red blood lysing buffer
1
Bone Marrow-Derived Dendritic Cell Culture
2
Quantifying GDNF and MSP Expression in Mitochondrial Disease
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Peripheral blood was collected from LT-hGDNF-2A-GFP- and LT-MSP-GFP-transplanted MitoPark mice and LT-MSP-GFP-transplanted non-MitoPark normal control mice. After erythrocytes were lysed with Red Blood Lysing Buffer (Sigma, St. Louis, MO, USA), the GFP-expressing (GFP+) cells were determined by flow cytometry (BD FACSCablibur System, BD Bioscience, San Jose, CA, USA). The peripheral blood samples collected from wild-type C57BL/6J and GFP transgenic mice were used as negative and positive controls, respectively.
3
Lung Tissue Isolation and Analysis
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At the endpoint of the experiment, all mice were euthanized by exposing them to overdose of isoflurane and then the lung tissues were removed for further analysis. Small portions of lung tissues were used in the isolation of bacterial DNA and histopathology, as described (Elliott, Nagarkatti, & Nagarkatti, 2016 ). Lung tissues were also smashed using a Stomacher 80 Biomaster lab blender (Metrohm USA, Riverview, FL) in 10 ml of RPMI‐1640 medium, lysed with Red Blood Lysing Buffer (Sigma‐Aldrich, St. Louis, MO, USA) to remove red blood cells, washed with PBS, filtered and resuspended in PBS supplemented with 5% FCS. Then lung‐associated mononuclear cells were isolated from lung cell suspensions in Ficoll‐Histopaque®‐1077 (Sigma‐Aldrich) at a 1:1 ratio under 350 g of centrifugation for 30 min, as described (Alghetaa et al., 2018 ). The lung‐associated mononuclear cells were stored immediately in TRIzol reagent at −80°C freezer for RNA sequencing. The portions of lung‐associated mononuclear cells were used in the measurement of proton efflux rate. Also, broncho‐alveolar lavage fluid (BALF) was collected from these euthanized mice according to the procedure described by Alghetaa et al. (2018 ).
4
Quantifying GFP Expression in Myeloid Cells
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Peripheral blood was collected from GFP-transplanted MitoPark mice at eight weeks-post BMT. After erythrocytes were lysed with Red Blood Lysing Buffer (Sigma, St Louis, MO), cells were labeled with APC-conjugated CD11b monoclonal antibody (BD Pharmingen, San Jose, CA) at a concentration of 0.4 µg per 106 cells and analyzed by flow cytometry (BD FACSCablibur System, BD Bioscience, San Jose, CA) for GFP expression. The peripheral blood samples collected from wild-type C57BL/6 J and GFP transgenic mice were used as negative and positive controls, respectively.
5
Dendritic Cell Generation from Mouse Bone Marrow
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Six week-old mice were used as a source of bone marrow (BM) for the generation of mouse DCs in culture. BM cells were isolated by flushing femurs with PBS. Pelleted cells were briefly resuspended in red blood lysing buffer (Sigma) to lyse red blood cells and stabilized by adding complete medium (RPMI 1640, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine). The cells were centrifuged and resuspended in complete medium supplemented with GM-CSF (100 ng/ml; Peprotech, NJ) to enhance replication of DCs [21] (link), [22] (link). Afterwards, cells were plated in non-tissue culture plastic Petri dishes (1 bone per 10 cm dish) for 5 days at 37°C with CO2.
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