Hepatocytes, non-parenchymal liver cells and liver macrophages were isolated as previously described with minor modifications [9 ,41 (link)]. In brief, livers were harvested, perfused with calcium and magnesium-free Hank's Balanced Salt Solution (HBSS; Beyotime Biotechnology, Shanghai, China) and digested with collagenase type I (Worthington, Lakewood, NJ, USA). The dissociated cells were filtered through a 70 μm cell strainer (Sorfa, Zhejiang, China) and then centrifuged at 50 g for 5 min at 4 °C. The hepatocyte pellets were collected and processed to eliminate erythrocyte using the Red Blood Cell Lysis Buffer (Beyotime Biotechnology). The supernatant fluids were transferred to a clean collection tube and centrifuged at 500 g for 10 min at 4 °C. The non-parenchymal liver cell pellets were collected and processed to eliminate erythrocyte using the Red Blood Cell Lysis Buffer (Beyotime Biotechnology). Liver macrophages were isolated from the non-parenchymal liver cell fraction as previously described with minor modifications [42 (link)]. Briefly, the non-parenchymal liver cell pellets were resuspended in sterile PBS (Sangon Biotech), gently layered on a double Percoll gradient (25% and 50%; GE Healthcare, Buckinghamshire, UK), and centrifuged at 900 g for 20 min at 4 °C. The layer between the 25%–50% gradient interface was collected.
Red blood cell lysis buffer
Manufactured by Beyotime
Sourced in China, United States
Red blood cell lysis buffer is a solution used to selectively lyse or break down red blood cells in a sample, while leaving other cell types intact. It is a core component in many cell isolation and purification protocols.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Axiovert 40 cfl phase contrast microscope, by Zeiss (3 mentions)
Guava easycyte, by Merck Group (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
M csf, by Thermo Fisher Scientific (3 mentions)
Cd11b, by BioLegend (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Collagenase 1, by Merck Group (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Hanks balanced salt solution (hbss), by Beyotime (3 mentions)
Phosphate buffered saline (pbs), by Beyotime (2 mentions)
Mouse regulatory t cell staining kit, by Thermo Fisher Scientific (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Brilliant stain buffer, by BD (2 mentions)
Fixable viability stain 780, by BD (2 mentions)
125 protocols using red blood cell lysis buffer
1
Isolation of Liver Cell Populations
2
Isolation of Luteinized Granulosa Cells and Follicular Fluid
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Thirty-four to 36 h after the administration of hCG, ovarian follicles were aspirated using a single-lumen, 17-gauge needle (Cook Medical, Bloomington, IN, USA) guided by trans-vaginal ultrasonography. Follicles were classified into two groups, the large follicle group with the diameter O18 mm and the small follicle group with diameter !10 mm as measured by ultrasonography. All the samples were collected by the same operator to ensure the size of the follicles. Large follicles were used to isolate luteinized granulosa cells. The oocytes were dissected from the collected follicles under a dissecting microscope. After collecting the oocytes, the remaining granulosa cells with fluid were transferred into sterile tubes (Axygen Scientific, Union City, CA, USA) and centrifuged at 200 g for 10 min. Afterwards, the supernatant was aspirated and collected as follicular fluid and cell pellets were washed with PBS (Beyotime, Shanghai, China) and subsequently a red blood cell lysis buffer (Beyotime) to eliminate red blood cells. The cells were stored at K80 8C or re-suspended for culture.
290 F Yang, Y-C Ruan and others
290 F Yang, Y-C Ruan and others
3
Establishment of LGG and GBM Cell Lines from Patient Tumor Samples
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Four low-grade glioma (LGG) and seven glioblastoma (GBM) specimens were derived from excess surgical materials of patients (Supplementary. Table S1 ). All of the patients had signed informed consent and were enrolled in accordance with the institutional protocols (Ethics number:2019-SR-479) approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. Patient-derived PL1 and PG7 cells were obtained from primary patient brain tumor specimens. In briefly, the divided tumor tissues were digested with 0.1% trypsin (Invitrogen, USA) and DNase I (Promega, USA) for one hour at 37 °C. Erythrocytes were lysed using Red Blood Cell Lysis Buffer (Beyotime, C3702, Shanghai, China). After being washed twice with PBS, the tissues were triturated by pipetting and passed through a 100-μm cell filter. PL1 and PG7 cells were cultured in DMEM containing 10% fetal bovine serum (Gibco, USA) at 37 °C with 5% CO2.
4
Quantifying Endothelial Progenitor Cells
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The co-expression of CD34, CD133, and Flk-1 (EPC markers) in implanted skin tissues or peripheral blood from mice was detected by flow cytometry. For isolation of single-cell suspensions, the skin tissues (1 cm × 0.5 cm) were grinded in pre-cooled phosphate buffer saline (PBS) and filtered through a nylon membrane (70 µm). After 5 min of centrifugation at 400 × g, the precipitated cells were washed with PBS for 3 times and then re-suspended in PBS containing 1% bovine serum albumin (BSA). Subsequently, the single-cell suspensions or blood samples were incubated with FITCconjugated anti-CD133 (BD Biosciences), Phycoerythrin (PE)-conjugated anti-Flk-1 (BD Biosciences), and PE-Cy5conjugated anti-CD34 (BD Biosciences) for 15 min in the dark. Red blood cells were removed using Red Blood Cell Lysis Buffer (Beyotime) for 5 min. Subsequently, the cells were washed, fixed with 2% paraformaldehyde, and kept in the dark at 4°C until fluorescence-activated cell sorter (FACS) analysis (BD Biosciences). EPC counts are expressed as a percentage of total cells in each sample. Flow cytometry was performed in a blinded fashion.
5
Tumor Immune Cell Isolation and Staining
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Tumors were harvested from both control and treatment groups. PBS was added to homogenize the tumor tissue into a single-cell suspension by passing a 70 μm strainer. Density-gradient centrifugation was performed using a Percoll centrifuge (Cytiva, 17089109) at 600 × g for 20 min at room temperature. Immune cells were carefully collected from the middle of the cloudy layer. After washing with PBS, the red blood cells were lysed using Red Blood Cell Lysis Buffer (Beyotime Company, C3702). Afterward, cells were incubated with eBioscience™ Brefeldin A (1000 × Solution, invitrogen, 00-4506-51) and eBioscience™ Monensin (1000 × Solution, invitrogen, 00-4505-51) for 4 h at 37℃ and 5 % CO2 to facilitate further intracellular proteins staining.
6
Quantitative Analysis of AMOG Silencing
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U-87 MG cells were seeded at a density of 1 × 104 cells per well in 8-well chamber slides or in 96-well cell culture plates in DMEM and 10% fetal bovine serum and allowed to settle overnight. They were then transfected with AMOG siRNA or control siRNA, and then assayed after 48 h for X-Gal staining by an overnight incubation at 37 °C, according to the manufacturer’s protocol (Beyotime Biotech, cat no. C0602). Images of 9 corresponding areas were captured light microscopically. For the measurement of β-galactosidase activity, cells were digested with red blood cell lysis buffer (cat no. C3702, Beyotime Biotech) and absorbance of X-Gal reaction product was measured with a microplate reader (Infinite M1000, Tecan, Männedorf, Switzerland).
7
Isolation and Culture of Murine Bone Marrow-Derived Macrophages
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Primary BMMs were isolated from the femurs and tibias of C57BL/6 mice. After dissecting aseptically under a laminar airflow hood, the ends of the bones were cut off with scissors and the marrow cells were flushed out using a sterile needle and syringe containing α-MEM. Red blood cells were removed by treatment with red blood cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). After washing, the cells were cultured in α-MEM containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2 in 60-mm cell culture dishes. After incubating overnight, the cell suspension was collected and re-cultured in a complete medium supplemented with 100 ng/mL M-CSF. After a 3-day culture, non-adherent cells were removed by washing with PBS and the adherent cells were cultured until they reached 90% confluence. In subsequent experiments, BMMs were counted and seeded at 5000 cells per well in 96-well plates and 10,000 cells per well in 48-well plates. To observe cell morphology, bright field images of monocytes were collected using a Zeiss Axiovert 40 CFL phase contrast microscope (Carl Zeiss Ltd., Oberkochen, Germany).
8
Isolation of Human Peripheral Blood Leukocytes
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Fresh anticoagulants from healthy people were collected and lysed by red blood cell lysis buffer (Beyotime Biotechnology, Shanghai, China) for several times until the supernatant became clear and transparent. The remaining cells were washed twice with PBS. The precipitates obtained were human peripheral blood leukocytes, which were used as the control group of AML cell lines.
9
Splenocyte Immunophenotyping Post-Treatment
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A total of 14 days post-treatment, spleens were excised from nine mice in each group, lysed with Red Blood Cell Lysis Buffer (Beyotime Institute of Biotechnology), centrifuged at 204 × g for 5 min at 4°C and washed with PBS. Cell suspension was prepared at a concentration of 1×107/ml. Subsequently, 100 µl cell suspension was incubated with appropriate fluorochrome-labelled CD3 (2 µl; cat. no. 100204), CD4 (1.5 µl; cat. no. 100412), CD8 (1.5 µl; cat. no. 100708) and natural killer (NK)1.1 (0.7 µl; cat. no. 108706) antibodies (all from BioLegend, Inc., San Diego, CA, USA) in the dark for 30 min at 4°C. Regulatory T cell (Treg) detection was performed using the Mouse Regulatory T Cell Staining kit (eBioscience; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The fluorescence intensity of cells was measured using a flow cytometer (Guava easyCyte; EMD Millipore) with a minimum of 10,000 cells collected.
10
Hydrogel-based Immunomodulatory Tissue Engineering
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GelMA and HAMA were synthesized in our laboratory. Bouin's solution was obtained from Fuzhou Phygene Biotechnology Co., LTD. Carboxyfluorescein diacetate succinimidyl ester (CFDA SE) cell proliferation assay and tracking kit, red blood cell lysis buffer, collagenase Ⅰ, collagenase Ⅱ, collagenase Ⅳ, hyaluronidase, cell counting kit 8 (CCK8), and Calcein AM/PI Live/Dead viability assay kit were bought from Shanghai Beyotime Biotechnology Co., Ltd. 1′-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR) was bought from Beijing Lablead Trading Co., LTD. Mouse TNF-α, mouse IL-6, and rat IgG ELISA kits were provided by Wuhan Elabscience Biotechnology Co., Ltd. aPD-1 (rat anti-mouse) and antibodies for flow cytometry used in this study were purchased from Biolegend Inc. (USA). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was obtained from Shanghai Aladdin Co., Ltd. CpG ODN 1826, with the sequence 5′-tccatgacgttcctgacgtt-3′ and a phosphorothioate backbone, and CPG ODN 1826 labeling with FAM on 3′ end (named FAM-CpG ODN) were obtained from Suzhou Synbio Technologies Co.
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