Lung tissue from euthanized post-natal 4 weeks SD rats were quickly extracted (trachea and excess tissue removed) and rinsed with PBS before being infiltrated in serum-free medium. Then we minced lung tissue into 1–3 mm pieces with sterile ophthalmic forceps in a dish and transferred pieces to serum-free medium containing Dispase II (Sigma-Aldrich, 4,942,078,001), DNase I (Sigma-Aldrich, D5025). After incubating at 37°C for 45 min with gentle shaking every 15 min, add 10% FBS to stop digestion. Digestion products passing through a 70 μm filter were centrifuged at 1500 rpm for 5 min. Added Red Cell Lysis Buffer (Beyotime, C3702) to resuspend, incubated at room temperature for 5 min, and then centrifuged at 1500 rpm for 5 min. After resuspending cells with DMEM with 10% FBS (Gibco, United States), 100 U/mL of penicillin and 100 μg/mL of streptomycin, PLFs were plated at a seeding density of 5 × 105–1 × 106/well with 2 mL complete medium in 6-well plate. All steps were performed on ice or at 4°C unless stated otherwise. Cells were cultured at 37°C in a 5% CO2 incubator with regular feeds and passaged at 80% fusion using 0.25% of trypsin–EDTA in a 1:3 ratio. PLFs were divided into three groups comprising a control group of cells without treatment, a group with TGFβ1 10 ng/mL treatment for 48 h, and a group with TGFβ1 10 ng/mL treatment for 72 h.
Red cell lysis buffer
Manufactured by Beyotime
Sourced in China
Red cell lysis buffer is a solution used to selectively lyse or break down red blood cells while preserving other cell types, such as white blood cells. It is a commonly used reagent in various laboratory procedures, including cell isolation, flow cytometry, and molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Macrophage colony stimulating factor, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Cd4 or cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Elispot kit, by R&D Systems (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
6 protocols using red cell lysis buffer
1
Isolation and Culture of Rat Pulmonary Fibroblasts
2
Immune Cell Profiling in Rat Model
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Rats were anesthetized. Blood samples (500 μl) were collected by cardiac puncture and put into a 2 ml centrifuge tube with anticoagulants. Red cell lysis buffer (Beyotime Biotechnology Co. Ltd., Jiangsu, China) were used following manufacturer’s instructions to remove red blood cells. The cells were stained by different antibodies for flow cytometry analysis (Cytomics FC 500, Beckman coulter), including CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), B lymphocytes (CD3+CD45RA+), NK cells (CD3-CD161a+), and monocytes (CD3-CD43+). For brain cell collection, rats were anesthetized and transcardially perfused with 0.9% NaCl. The ischemic hemisphere was dissected. The cells were isolated from brain tissues for flow analysis on a BD flow cytometer using FACS Diva 6.0 software. The data were analyzed by FlowJo (BD, USA).
3
Isolation and culture of mouse BMDMs
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Eight-week-old BKS-DB female mice were used to obtain BMDMs, which were flushed by DMEM through the femur and tibia with a 25-gauge needle. The cell suspension was filtered using 70 μm cell filters and centrifuged at 1,000 rpm for 5 min. The cell pellet was subsequently resuspended in red cell lysis buffer (Beyotime, China) for 3 min. After being centrifuged and resuspended, cells were cultured in DMEM containing 1% L-glutamine, 30 mM glucose, 10% fetal bovine serum (FBS, ThermoFisher Scientific), 1% penicillin‒streptomycin (Sigma‒Aldrich) and 20 ng/mL macrophage colony stimulating factor (PeproTech). On day 3, half of the medium was replaced, and on day 5, all medium was replaced with fresh medium. On day 7, BMDMs were used for related experiments. Mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 30 mM glucose, 10% FBS and 1% penicillin‒streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator.
4
Cytotoxic T Cell Response Analysis
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The 108 GL261 cells were collected and resuspended in DMEM. These cells were then frozen in a −80°C freezer for 1 h and thawed at 37°C for three cycles to lyse the cells. The solution was centrifuged at 300 rpm for 30 min, and the supernatants were collected and stored at 4°C for further use as tumor-specific antigens.
The mice were euthanized, and the spleens were collected. The spleens were disassociated by squeezing, and all released and suspended cells were collected. The suspended cells were treated with red cell lysis buffer (Beyotime), and the splenocytes were centrifuged at 400 g for 3 min. The splenocytes were cultured with GL261 cell lysates (1:3 for the cell numbers) for 48 h. The lymphocytes were isolated from cultured splenocytes using gradient centrifugation on histopaque-1077. CD4+ T and CD8+ T cells were isolated from lymphocytes according to the instructions of CD4+ or CD8+ T cell isolation kits (Miltenyi Biotec).
GL261 cells expressing a control shRNA or H2-KD shRNA (MHC-I subunit) were infected with lentivirus expressing PTEN shRNA. CD4+ or CD8+ T cells were co-cultured with GL261 cells at a ratio of 3:1 for 24 h, and CD4+ or CD8+ T cells were then collected and subjected to ELISpot analysis with an ELISpot kit (R&D Systems) containing plates coated with an anti–IFNγ or an anti–IL-2 antibody.
The mice were euthanized, and the spleens were collected. The spleens were disassociated by squeezing, and all released and suspended cells were collected. The suspended cells were treated with red cell lysis buffer (Beyotime), and the splenocytes were centrifuged at 400 g for 3 min. The splenocytes were cultured with GL261 cell lysates (1:3 for the cell numbers) for 48 h. The lymphocytes were isolated from cultured splenocytes using gradient centrifugation on histopaque-1077. CD4+ T and CD8+ T cells were isolated from lymphocytes according to the instructions of CD4+ or CD8+ T cell isolation kits (Miltenyi Biotec).
GL261 cells expressing a control shRNA or H2-KD shRNA (MHC-I subunit) were infected with lentivirus expressing PTEN shRNA. CD4+ or CD8+ T cells were co-cultured with GL261 cells at a ratio of 3:1 for 24 h, and CD4+ or CD8+ T cells were then collected and subjected to ELISpot analysis with an ELISpot kit (R&D Systems) containing plates coated with an anti–IFNγ or an anti–IL-2 antibody.
5
Bronchoalveolar Lavage Fluid Analysis
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BALF was obtained by inserting a 20-gauge catheter into the trachea through which 1 ml of cold PBS was flushed back and forth 3 times. BALF was centrifuged at 1500 rpm for 10 min at 4 °C. Cell-free supernatants were used for the measurement of total protein concentration with a BCA protein assay kit (Thermo Fisher Scientific Inc, Waltham, MA USA). The BALF cell pellet treated with red cell lysis buffer (Beyotime Inc, Jiangsu, China) was re-suspended in PBS for cell count and immuno-phenotyping.
6
Xenograft Tumor Monitoring in NOG Mice
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Xenograft studies were conducted on NOG mice (Shanghai Model Organisms Center, Inc.), with >6 mice in each group. The mice were intravenously injected with 1 × 106 Nalm6 cells on day 0, and with 1 × 106 CAR-T cells and NT-T cells on day 7.
At the end of every seven days of Nalm6 injection, 50 μL of the peripheral blood was collected and processed to remove the red blood cells using red cell lysis buffer (Beyotime, Shanghai, China). The T cells and Nalm6 cells were stained with anti-human CD19 and CD3 antibodies. The counts of T cells and Nalm6 cells were analyzed by the CountBrightTM Absolute Counting Beads (C36950, Thermo Fisher Scientific), as suggested by the manufacturer.
At the end of every seven days of Nalm6 injection, 50 μL of the peripheral blood was collected and processed to remove the red blood cells using red cell lysis buffer (Beyotime, Shanghai, China). The T cells and Nalm6 cells were stained with anti-human CD19 and CD3 antibodies. The counts of T cells and Nalm6 cells were analyzed by the CountBrightTM Absolute Counting Beads (C36950, Thermo Fisher Scientific), as suggested by the manufacturer.
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