Following euthanasia, the long bones of the animals were isolated. Bone marrow was flushed with sterile PBS. Red blood cells (RBCs) were lysed using RBC lysis buffer (Solarbio). Equal cell numbers were cultured overnight in 50 ng/mL M-CSF (BioLegend) for 24 h to obtain bone marrow-derived monocytes/macrophages (BMMs). The suspension cells were cultured in α-Minimal Essential Medium (αMEM) containing 10% FBS and 1% penicillin‒streptomycin solution with M-CSF (50 ng/mL) and RANKL (100 ng/mL). The CM treatments were 1:1 with αMEM with 10% FBS. To detect the effects of cytokines on the expression of GPR84 in BMMs, CTGF (50 ng/mL), IL-11 (10 ng/mL), PTHrP (100 nM) and EGF (10 ng/mL) were added. For treatment with agonists, cells were seeded in 24-well plates and treated with 6-OAU (200 nM) for 6 h. To activate the MAPK pathway, 50 μM tert-butylhydroquinone (t-BHQ) or 10 μM anisomycin was added to the culture medium for 6 h. In some experiments, cells were transfected with pCMV6-GPR84 for 24 h. Then, osteoclast differentiation was carried out for two to four days, with the differentiation medium being replaced on day three. For tartrate-resistant acid phosphatase (TRAP) staining, cells were fixed in 4% paraformaldehyde (PFA) for 30 min and then stained with TRAP staining solution (Wako, Japan) according to the manufacturers’ instructions.
Rbc lysis buffer
Manufactured by Solarbio
Sourced in China
RBC lysis buffer is a solution used to selectively lyse (break down) red blood cells (RBCs) in biological samples. It effectively removes RBCs, allowing for the isolation and analysis of other cell types, such as leukocytes (white blood cells), from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Collagenase type 4, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Histopaque 1083, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Trap staining solution, by Fujifilm (1 mentions)
M csf, by BioLegend (1 mentions)
Staining buffer, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse cd4, by BioLegend (1 mentions)
Apc conjugated anti mouse b220, by BioLegend (1 mentions)
Pacific blue conjugated anti mouse gl 7, by BioLegend (1 mentions)
Pe conjugated anti mouse cd95, by BioLegend (1 mentions)
Pe conjugated anti mouse pd 1, by BioLegend (1 mentions)
38 protocols using rbc lysis buffer
1
Osteoclast Differentiation from Bone Marrow-Derived Monocytes
2
Spleen Cell Immunophenotyping by Flow Cytometry
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The mice were humanely sacrificed, and the spleens were removed. The spleens were triturated, and the red blood cells were removed using RBC Lysis Buffer (Solarbio) according to the kit instructions. After centrifuging at 500 g for 10 min at 4°C, the supernatants were discarded, and cells were resuspended and washed twice with Staining buffer (eBioscience) to obtain single-cell suspensions. Then, cells were stained with different combinations of flow cytometry antibodies, including APC-conjugated anti-mouse CD3 (eBioscience, San Diego, United States), FITC-conjugated anti-mouse CD4 (BioLegend, San Diego, United States), PE-conjugated anti-mouse CD8 (eBioscience) APC-conjugated anti-mouse B220 (BioLegend), Pacific Blue-conjugated anti-mouse GL-7 (BioLegend), PE-conjugated anti-mouse CD95 (BioLegend), PE-conjugated anti-mouse PD-1 (BioLegend), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (BioLegend). After staining, cells were washed with Staining buffer and dispersed in 500 mL of Staining buffer. Analysis was performed using a Mona CytoFLEX flow cytometer (BeckmanCoulter LifeSciences, Brea, United States).
3
Single-Cell Isolation from Gastric Cancer
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Fresh tumor tissue samples were obtained from 5 GC patients by endoscopic ultrasonography-guided biopsy from surgery before and after treatment. Tissue was immediately immersed in RPMI 1640 medium (Thermo, 11875-085) for subsequent single-cell isolation. Tissue dissociation was performed with Tumor Dissociation Kit (Miltenyi Biotec, 130-095-929) at 37°C for 30-45 min and filtered using a 40μm cell strainer (BD, 352340). Erythrocytes were removed by RBC lysis buffer (Solarbio, R1010). Cell suspensions were washed 2 times with PBS containing 0.04% BSA at 300g for 5 min at 4°C. Cell viability was measured with Trypan Blue (Thermo fisher, 15250061) staining. Cell concentration was measured with hemocytometer and adjusted to 700-1,500 cells/μL.
4
Isolation of high-grade serous ovarian cancer cells
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Tissue samples were collected from three patients diagnosed with high-grade serous OC at Women’s Hospital, Zhejiang University School of Medicine, China. The study was approved by the Hospital Ethical Committee (approval no. IRB-20220229-R). The samples were surgically removed and immediately processed as the following steps. Briefly, tissue samples were cut into several small pieces on ice and then digested using a mix of collagenase I, IV and DNase I (Sigma). Next, digested samples were filtered with a 40-μM cell strainer and then harvest the cells by centrifuging. After lysing the red blood cells (RBC) by RBC lysis buffer (Solarbio, R1010), the cells were harvested finally and resuspended in Advanced DMEM/F12 medium (Gibco, 12634028) containing 10% FBS.
5
Tracking AML Cell Targeting by Ara-C@HFn
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The AML-bearing mouse model was established as described in a previously published paper [17 (link)]. Briefly, SCID mice were injected with cyclophosphamide (100 mg/kg) intraperitoneally and inoculated with 5 × 106 AML cells intravenously the following day. At 15 days post HL-60 cell injection, the mice were injected with Cy5-labeled Ara-C@HFn, and 2 h later, the peripheral blood, spleen and hindlimbs of the AML-bearing mice were collected to evaluate targeting capacity. Then, the spleen and hindlimbs were used to prepare single-cell suspensions, red blood cells (RBCs) were removed from the peripheral blood and single-cell suspensions by using RBC lysis buffer (Solarbio). Subsequently, these samples were incubated with EV450-conjugated anti-human CD45 (anti-hCD45) antibody (eBioscience) for 30 min, and the proportion of hCD45+ cells (the marker of HL-60 cells) in Cy5+ (the label of Ara-C@HFn) cells was analyzed by flow cytometry (n = 3).
6
Lung Immune Cell Profiling by Flow Cytometry
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For flow cytometry, lungs were harvested on days 21. Lungs were cut into small pieces and digested in 5 mL of digestion buffer consisting of RPMI-1640 (Biological Industries), collagenase IV (1.6 mg/mL, Worthington Biochemical Corp), and DNase1 (50 unit/mL, Worthington Biochemical Corp). Lungs were shaken at 37 °C for 30 min, and RBCs were lysed using RBC lysis buffer (Solarbio). Homogenized lung was passed through a 70 μm cell strainer (Biologix) to obtain a single-cell suspension. Cells were washed twice with cold PBS and centrifuged at 300 g for 8 min at 4 ℃. 1 × 106 cells were resuspended in 100 µL of cold PBS per sample and stained with APC-conjugated anti-LY6C1 (Thermo, 17-5932-82) and anti-CCR2 (Abcam, ab216863). Primary antibody CCR2 further incubated with Cy3-conjugated goat anti-rabbit secondary antibody (Abcam, ab97075). One hundred thousand events per sample were collected (BD, FACS Canto II), and data were analyzed with FlowJo V10.5.3 software. Cell gating was based on the comparison with isotype control.
7
Vitamin C Modulates Cellular Responses
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Vitamin C (purity: > 99.9%) was purchased from Shandong Xinhua pharmaceutical CO., ltd. DMEM Medium, Fetal Bovine Serum (FBS), Wright-Giemsa staining, and Albumin Bovine V were purchased from MACGENE. RBC lysis Buffer, Sulforhodamine B (SRB), RNase A, Propidium Iodide (PI), and rat IgG were purchased from Solarbio. Triton X-100 solution, Ca2+ specific fluorescent probe Fluo-4/AM, JC-1 Staining Kit, One Step TUNEL Apoptosis Assay Kit, and ATP Determination Kit were purchased from Beyotime. FITC Rat Anti-Mouse CD11b was purchased from BD Biosciences. APC Rat Anti-Mouse CD206 was purchased from Miltenyi Biotec. Western blot Antibody Diluent was purchased from Epizyme. Western Bright™ ECL and Western Bright™ Peroxide were purchased from Advansta. Goat Anti-Rabbit IgG (H+L) HRP was purchased from Affinity Biosciences. Vimentin (5G3F10) Mouse mAb, Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate) and β-actin (13E5) Rabbit mAb were purchased from Cell Signaling Technology. E-cadherin Rabbit PolyAb was purchased from Proteintech.
8
CD47-targeting siRNA Transfection Protocol
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Dulbecco’s modified Eagle’s medium (DMEM), PBS, penicillin/streptomycin, l-glutamine, fetal bovine serum (FBS), Aqua Dead Cell Stain Kit, Lipofectamine 2000, TRIzol, and collagenase type IV were purchased from Thermo Fisher Scientific (Waltham, MA, United States). The RBC lysis buffer was purchased from Solarbio (Beijing, China). siRNA-targeting mouse CD47 mRNA (antisense strand, 5’-UGGUGAAAGAGGU-CAUUCCdTdT-3’) and negative control siRNA with a scrambled sequence (antisense strand, 5’-ACGUGACACGUUCGGAGAAdTdT-3’) were synthesized by Suzhou Biosyntech Co. Ltd. (Suzhou, China). Anti-CD47 antibody was purchased from Santa Cruz Biotechnology (Texas, United States). The Click-iT Plus TUNEL Assay was performed for in situ apoptosis detection; the Alexa Fluor 647 dye was purchased from Thermo Fisher Scientific (MA, United States).
9
Splenic B Cell Isolation and Culture
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Splenocytes were prepared by lightly disrupting 70 μm cell filters of the spleen. Erythrocytes were lysed with RBC lysis buffer (Solarbio, China). The splenocytes were subjected to magnetic bead sorting, using CD19 antibody-biotin (Miltenyi Biotec, Germany) and Anti-biotin microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s recommendations, to obtain purified splenic B cells. Cells were then cultured in 1,640 medium containing 10% FBS, anti-IgM (10 μgmL−1, Thermo, United States), CD40L (100 ng mL−1, RD, United States) and IL-4 (20 ng mL−1, RD, United States), 100 U/ml penicillin and streptomycin in 5% CO2.
10
Quantification of Murine Leukocyte Subsets
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Leukocytes from mice blood, spleen, and bone marrow (BM) were quantified using flow cytometry. Briefly, 100 μL peripheral blood was collected in EDTA anticoagulant tubes. Single-cell suspensions of the spleen were obtained through mechanical disaggregation. Single-cell suspensions of the BM were flushed from femurs and tibias. These single-cell suspensions were then filtered through 70 μm cell strainers (15-1070, Biologix, West Hollywood, CA, USA). The erythrocytes were lysed using RBC lysis buffer (R1010, Solarbio, Beijing, China). After 2 washes with staining buffer, cells were preincubated with Human TruStain FcX™ (422302, Biolegend, San Diego, CA, USA) for 10 min at room temperature, followed by staining with fluorochrome-labeled antibodies (1:50) in the dark at 4 °C for 45 min, and detection with an LSRfortessa flow cytometer (BD Biosciences, San Jose, CA, USA). The following fluorochrome-conjugated antibodies were used: PE-Cy™5 anti-human CD45 (555490, BD Biosciences), PE anti-mouse CD45 (561087, BD Biosciences), APC R700 anti-human CD3 (659119 BD Biosciences), APC anti-human CD4 (565994, BD Biosciences), FITC anti-human CD8 (555634, BD Biosciences), BV421 anti-human CD19 (562440, BD Biosciences). The data were analyzed employing FlowJo software.
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