Expression of identified DECRs was validated further in clinical samples by RT-qPCR. RNA isolation and RT-qPCR were undertaken as described previously (Zhou et al., 2021 (link)). In brief, red blood cell (RBC) lysis buffer (catalog number: C3702; Beyotime Institute of Biotechnology, Shanghai, China) was used to split RBCs. Subsequent extraction of total RNA by TRIzol® Reagent and synthesis of complementary-DNA were carried out according to manufacturer (Takara Biotechnology, Shiga, Japan) protocols. mRNA expression was quantified using the ABI-9700 system (Applied Biosystems, Foster City, CA, United States) and normalized against the housekeeping gene β-actin by the comparative quantification method (2−ΔΔCT). The primers used in this study are listed in Supplementary Table S2 .
Rbc lysis buffer
Manufactured by Beyotime
Sourced in China
RBC lysis buffer is a solution used to selectively lyse (break down) red blood cells (RBCs) in a sample, while leaving other cell types, such as white blood cells, intact. This allows for the isolation and analysis of the remaining cell populations.
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Lab products found in correlation
Cytoflex, by Beckman Coulter (4 mentions)
Dnase 1, by Merck Group (3 mentions)
Collagenase 2, by Merck Group (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Hyaluronidase, by Solarbio (2 mentions)
Debris removal solution, by Miltenyi Biotec (2 mentions)
Trizol reagent, by Takara Bio (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Cd11b, by BD (1 mentions)
Macs dissociator, by Miltenyi Biotec (1 mentions)
Mouse il 6 and mcp 1 elisa kits, by Thermo Fisher Scientific (1 mentions)
Tetrachloroauric acid trihydrate, by Merck Group (1 mentions)
Murine gm csf, by Thermo Fisher Scientific (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Pe cy7 anti mouse cd105, by BioLegend (1 mentions)
25 protocols using rbc lysis buffer
1
Validating DECR Expression in Clinical Samples
2
Lung Cell Isolation Protocol
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After BAL, lungs were dissected into pieces for enzyme digestion in RPMI-1640 medium containing DNase I (0.15 mg/mL, Sigma, USA) and collagenase II (1 mg/mL, Sigma, USA) at 37 ℃ for 1 h [27 (link), 28 (link)]. The digested tissues were homogenized by vigorous shaking and passed through a 70-μm cell strainer (BD, USA). The cell suspensions were lysed with RBC Lysis Buffer (Beyotime, Shanghai, CHN) to remove red blood cells, and stained for flow cytometry analysis.
3
Isolation of Mouse Peritoneal Macrophages
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The setup of mouse experiments was approved by the institutional regulation of Model Animal Research Center of Soochow University (Suzhou, China). Operation and termination of mice were performed according to the criteria of the animal welfare office of Soochow University.
Macrophages from mouse were isolated at the age of 6 weeks. Mice were injected with 1 ml liquid paraffin intraperitoneally 3 days before isolation according to Ray’s method [13 ]. At the time of isolation, mice were euthanized and sterilized with 70% ethanol. It was immediately followed by the intraperitoneally injection of 5 ml cold RPMI using a 27 g needle. After gently massage the peritoneum, ascitic fluid was collected using a 25 g needle. Peritoneum was opened by incision and the remaining fluid was collected by Pasteur pipette. Cell suspension was washed once with cold PBS and treated with RBC lysis buffer (Beyotime, Shanghai, China) for 1 min to get rid of erythrocytes. Three hours after seeding, the adherent cells were gently washed once with PBS and cultured in full RPMI medium for further analysis. Enriched macrophages were identified and counted by flow cytometry (CytoFLEX, Beckman Coulter, CA, USA).
Macrophages from mouse were isolated at the age of 6 weeks. Mice were injected with 1 ml liquid paraffin intraperitoneally 3 days before isolation according to Ray’s method [13 ]. At the time of isolation, mice were euthanized and sterilized with 70% ethanol. It was immediately followed by the intraperitoneally injection of 5 ml cold RPMI using a 27 g needle. After gently massage the peritoneum, ascitic fluid was collected using a 25 g needle. Peritoneum was opened by incision and the remaining fluid was collected by Pasteur pipette. Cell suspension was washed once with cold PBS and treated with RBC lysis buffer (Beyotime, Shanghai, China) for 1 min to get rid of erythrocytes. Three hours after seeding, the adherent cells were gently washed once with PBS and cultured in full RPMI medium for further analysis. Enriched macrophages were identified and counted by flow cytometry (CytoFLEX, Beckman Coulter, CA, USA).
4
Isolation of Immune Cells from Tissues
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Spleens, Peyer’s patches (PPs) and MLNs were collected on sacrifice under sterile conditions. Single cell suspensions were prepared from spleen, PP, and MLN by pressing a piston and through a cell strainer, and the collected cells were washed with PBS. To isolate splenocytes, red blood cells were removed by RBC lysis buffer (Beyotime, Jiangsu, China). Then the lymphocytes and splenocytes were used as starting material for further analyses including RT-qPCR, ELISA, and flow cytometry.
5
Isolation and Culture of Mouse Bone Marrow Cells
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Male C57BL/6 mice (six-week-old male, 18-20 g) were sacrificed by cervical dislocation under anesthetization and disinfected with 75% alcohol for 3-5 min. The femurs and tibiae were carefully isolated and flushed with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA). Cells were centrifugated at 300 ×g for 10 min, and re-suspended using red blood cell (RBC) lysis buffer (500 μl, Beyotime, China) for 10 min. Cells were centrifugated at 300 ×g for 10 min, and were re-suspended using PBS. Cells were re-suspended in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco) in culture flasks at a density of 1 × 105 cells/ml. Adherent cells were cultured by changing the medium every 3 days and the cells were used for subsequent experiments following 3 passages.
6
Isolation of Mouse Brain Cells
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For isolation of cells, fresh mouse brain samples were cut into pieces and digested in DMEM supplemented with collagenase IV (1 mg/ml, Gibco), DNase I (20 U/ml, Sigma-Aldrich) and hyaluronidase (0.01%, Solarbio) for 30 min at 37 °C. After digestion, the cells were filtered through a 70-μm strainer, and Debris Removal Solution (130-109-398, Miltenyi Biotec) was applied to remove myelin according to the manufacturer’s instructions. Cell pellets were then treated with RBC lysis buffer (C3702, Beyotime) and resuspended in FACS staining buffer (PBS containing 2% FBS).
7
Isolation and Characterization of Bone Marrow Cells
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Femur samples were collected, bone marrow was extracted, single cells were collected, and they were centrifuged at 1500 rpm for 5 min at 4 °C, after which the supernatant was discarded. The pellet was lysed in 1 × RBC lysis buffer (Beyotime, Shanghai, China) for 15 min. The surface markers of each cell type were quantified by flow cytometry, and the number of cells used for analysis was 1 × 106/tube.
Monoclonal antibodies FITC-Lin-, APC-c-kit, and PE-sca1 were used for the detection of LSK cells in bone marrow. Monoclonal antibodies against PE-Ly6C and FITC-F4/80 were used to detect changes in the number of monocytes and macrophages in mouse bone marrow; the flow antibody list is shown in theTable 4 . The 1 × 106 cells were washed twice with phosphate buffered saline (PBS), and resuspended in 100 µL PBS containing monoclonal antibodies and incubated at 4 °C for 30 min. The cells were then washed twice and resuspended in 200 µL PBS. A flow cytometer (Aria II, USA) was used for fluorescence analysis. Percentages of positive cells were evaluated based on fluorescence intensity.
Monoclonal antibodies FITC-Lin-, APC-c-kit, and PE-sca1 were used for the detection of LSK cells in bone marrow. Monoclonal antibodies against PE-Ly6C and FITC-F4/80 were used to detect changes in the number of monocytes and macrophages in mouse bone marrow; the flow antibody list is shown in the
8
Bronchoalveolar Lavage Cell Analysis
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After complete anesthesia with 0.2 ml 1% sodium pentobarbital (100 mg/kg, ip), bronchoalveolar lavage was performed with 2 ml sterile PBS via an endotracheal tube. The bronchoalveolar lavage fluids (BALF) were centrifuged at 4 ºC, 1000 rpm for 10 min. Cell pellets were re-suspended in PBS with Red Blood Cell (RBC) Lysis Buffer (C3702, Beyotime technology, Shanghai, China), for total leukocytes counting using a hemocytometer. Then, smeared BAL cells were stained with Wright-Giemsa stain solution (Baso Diagnostics Inc, Zhuhai, China).
9
Isolation and Cultivation of Rat Peritoneal Macrophages
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Rats were anesthetized by intravenous (i.v.) administration of pentobarbital sodium (50 mg/kg BW). Rat peritoneal macrophages were harvested by sterile lavage of the peritoneal cavity with 20 mL cold RPMI medium (without fetal bovine serum and antibiotics, Gibco, USA). After 10 min, the lavage fluid was collected and centrifuged (1,200 × g, 6 min). Supernatant was removed and red blood cell (RBC) lysis buffer (Beyotime, China) was added. After 3 min, the cell suspension was centrifuged (1,200 × g, 3 min), the supernatant decanted, and the cells washed with cold phosphate buffered saline (PBS, pH 7.4). The cells were then resuspended in RPMI 1640 supplemented with 10% FBS, 100 ng/mL penicillin, and 100 ng/mL streptomycin, and incubated at 37 °C for 2 h. Nonadherent cells were then removed by washing with PBS. The adherent macrophages were used for protein extraction.
10
Isolation and Phenotyping of Murine Omental Macrophages
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Murine omental tumor and omentum tissues were mechanically minced with scissors into small pieces, followed by digestion at 37 °C in a 5% CO2 incubator for 30 min in RPMI 1640 supplemented with 10% FBS, 1% penicillin–streptomycin, 0.05 mg/ml collagenase type I (Sigma-Aldrich), 0.025 mg/ml hyaluronidase (Sigma-Aldrich), and 0.01 mg/ml DNase I (Roche). Digested samples were passed through a 70-μm mesh, and red blood cells were lysed in RBC lysis buffer (Beyotime) for 3 min. Cell labeling was performed using fluorescently conjugated antibodies directed against mouse CD45, CD11b, F4/80, and CD163. Flow cytometry was performed on a DxFLEX flow cytometer (Beckman), and subsequent data were analyzed using FlowJo V10.
M0 macrophages derived from THP-1 cells and peritoneal macrophages were washed, resuspended, and stained with anti-CD163 antibody. Finally, the cells were analyzed using flow cytometry. The antibodies used for flow cytometry analyses are listed in Supplemental TableS3 .
M0 macrophages derived from THP-1 cells and peritoneal macrophages were washed, resuspended, and stained with anti-CD163 antibody. Finally, the cells were analyzed using flow cytometry. The antibodies used for flow cytometry analyses are listed in Supplemental Table
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