The FCA samples of mouse spleen were prepared as previously reported [16 (link)]. In brief, mouse spleen tissues were ground in PBS and then passed through a 70 μm filter. Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cells, followed by resuspension of the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy™7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy™7 Rat anti-mouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
R1010
Manufactured by Solarbio
Sourced in China
The R1010 is a compact and reliable lab equipment designed for general laboratory use. It serves as a platform for various experiments and procedures, providing a stable and controlled environment for sample handling and analysis. The core function of the R1010 is to assist researchers and technicians in their day-to-day laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Percp cyanine5.5 anti mouse cd4, by BioLegend (2 mentions)
Pe rat anti mouse f4 80, by BD (2 mentions)
Fitc hamster anti mouse cd3e, by BD (2 mentions)
Apc anti mouse cd11c, by BioLegend (2 mentions)
M csf 216 mc 025, by R&D Systems (2 mentions)
Apc cy7 rat anti mouse cd45, by BD (1 mentions)
Pe cy7 rat anti mouse cd8a, by BD (1 mentions)
Compensation beads, by BD (1 mentions)
True nuclear transcription factor buffer set, by BioLegend (1 mentions)
Tcrγ δ, by BioLegend (1 mentions)
Cd16 32, by BioLegend (1 mentions)
Granzyme b, by BioLegend (1 mentions)
Cd11b, by BioLegend (1 mentions)
F4 80, by BioLegend (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
P9040, by Solarbio (1 mentions)
Penicillin streptomycin c0222, by Beyotime (1 mentions)
Facscanto 2, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
25 protocols using r1010
1
Flow Cytometry Analysis of Mouse Spleen Cells
2
Immunophenotyping of Mouse Splenic Leukocytes
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The FCA samples of mouse spleen were prepared as previously reported [19] . In brief, mouse spleen tissues were ground in PBS and then passed through a 70 μm filter.
Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cell, followed by re-suspending the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy TM 7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy TM 7 Rat antimouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cell, followed by re-suspending the spleen sample with PBS. The prepared single-cell suspension from spleen was stained with FVS (564997, BD Pharmigen, San Diego, CA, USA), APC-Cy TM 7 Rat anti-mouse CD45 (557659, BD), PE Rat anti-mouse F4/80 (565410, BD), APC anti-mouse CD11c (117310, Biolegend, San Diego, CA, USA), FITC Hamster anti-mouse CD3e (553061, BD), PerCP/Cyanine5.5 anti-mouse CD4 (100434, Biolegend) and PE-Cy TM 7 Rat antimouse CD8a (552887, BD) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (BD Bioscience, San Diego, CA, USA).
3
Isolation of Neutrophils from Peripheral Blood
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Peripheral whole blood was taken from healthy adults and anticoagulated with EDTA. Neutrophils were isolated using Polymorphprep (AS1114683, Oslo, Norway) and centrifuged at 500g for 30 minutes, following the isolation procedure as instructed. Carefully extract neutrophils, wash twice with PBS resuspension, and centrifuge at 400g for 10 minutes. Add erythrocyte lysis solution (R1010, Solarbio, China) and lyse at 4°C for 15 minutes, centrifuge and discard supernatant until no red color is present in the precipitate. We use a cell counter to count the number of cells. Healthy adults were recruited from March 1 to April 1, 2023, and all received written informed consent. The first affiliated Hospital of Gannan Medical University’s Institutional Review Board and Ethics Committee approved the study (Ganzhou China, ethical approval No. LLSC-2023022801).
4
Isolation of Bovine Mononuclear Cells
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Density gradient separation was used to isolate mononuclear cells from blood. Holstein cows were bled by coccygeal venipuncture and blood were collected in 20 mL Sodium heparin anticoagulation tube (Junnuo, Shandong, China). Samples were then diluted in an equal amount of phosphate buffer saline (PBS) containing 0.02% ethylene diamine tetraacetic acid (EDTA), layered on Biocoll Separating Solution before centrifuging at 800 × g for 30 min. The interphase containing mononuclear cells was collected according to manufacturer's instructions (P5280, Solarbio, Beijing, China) and shaken for 40 s to lyse erythrocytes (R1010, Solarbio) (250 x g for 10 min). Then, mononuclear cells were washed twice with PBS containing 5 g/L of bovine serum albumin (BSA) and 2.0 mM EDTA (250 × g for 10 min).
5
Isolation and Characterization of Bone Marrow-Derived Macrophages
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BMDMs were isolated from the femur and tibia of WT and Usp25-/- mice. Briefly, bones were flushed with RPMI 1640 containing 100 U/mL penicillin and 100 μg/mL streptomycin. Then, samples were filtered using a 70-mm nylon mesh and collected in 50-mL tubes. Red blood cell lysis was performed (R1010; Solarbio, Beijing, China) and samples were spun at 1000 × g for 5 minutes. Cells were cultured in 20% L929 cell culture medium. On days 3 and 5, fresh Dulbecco’s modified Eagle medium (DMEM) containing 20% L929 cell culture medium and 10% fetal bovine serum was added. Cells were used for experiments on day 7. Studies included exposure of BMDMs to the following: acinar cell supernatant to assess activation, STING agonist DMXAA for signaling pathway determination, and IL17A to assess crosstalk between signaling axes. After exposure of BMDMs to acinar cell supernatant, levels of IFNβ were detected by ELISA.
For some studies, BMDMs isolated from Usp25-/- mice were transfected with control (B21030500; Genechem, Shanghai, China) or plasmid expressing Flag-tagged Usp25 (NM_013918; Genechem).
For some studies, BMDMs isolated from Usp25-/- mice were transfected with control (B21030500; Genechem, Shanghai, China) or plasmid expressing Flag-tagged Usp25 (NM_013918; Genechem).
6
Multicolor Flow Cytometry Analysis of Immune Cells
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The colon and rectum with adenoid tumor were collected and digested with the Lamina Propria Dissociation Kit, mouse (130-097-410), according to the manufacturer's direction.
The spleens were ground into single cells and lysed with red blood cell lysis buffer (R1010, Solarbio) on ice after centrifugation; the followed steps were done according to the instruction. All the procedures were carried out according to BD procedure steps with anti-mouse antibody for CD45 (103111/103137, BioLegend), CD3 (100203, BioLegend), CD4 (100407, BioLegend), CD8a (100709, BioLegend), TCRγ/δ (118129, BioLegend), NK1.1 (108713, BioLegend), F4/80 (123107, BioLegend), FOXP3 (126419, BioLegend), CD25 (101917, BioLegend), granzyme B (372216, BioLegend), CD16/32 (101319, BioLegend), CD11b (101208, BioLegend), CD11c (1173229, BioLegend), Ly-6C (128017, BioLegend), and Ly-6G (127613, BioLegend). After being blocked with TruStain FcX (anti-mouse CD16/32) antibody, cells were stained with antibodies for cell surface markers and then the intracellular biomarker. True-Nuclear™ Transcription Factor Buffer Set (424401, BioLegend) and fixation buffer (BioLegend, 420801) were used according to the protocol recommended by BioLegend. Before that, BD Compensation Beads (BD Biosciences, 552845) were used to make the best fluorescence compensation settings for multicolor flow cytometric analysis.
The spleens were ground into single cells and lysed with red blood cell lysis buffer (R1010, Solarbio) on ice after centrifugation; the followed steps were done according to the instruction. All the procedures were carried out according to BD procedure steps with anti-mouse antibody for CD45 (103111/103137, BioLegend), CD3 (100203, BioLegend), CD4 (100407, BioLegend), CD8a (100709, BioLegend), TCRγ/δ (118129, BioLegend), NK1.1 (108713, BioLegend), F4/80 (123107, BioLegend), FOXP3 (126419, BioLegend), CD25 (101917, BioLegend), granzyme B (372216, BioLegend), CD16/32 (101319, BioLegend), CD11b (101208, BioLegend), CD11c (1173229, BioLegend), Ly-6C (128017, BioLegend), and Ly-6G (127613, BioLegend). After being blocked with TruStain FcX (anti-mouse CD16/32) antibody, cells were stained with antibodies for cell surface markers and then the intracellular biomarker. True-Nuclear™ Transcription Factor Buffer Set (424401, BioLegend) and fixation buffer (BioLegend, 420801) were used according to the protocol recommended by BioLegend. Before that, BD Compensation Beads (BD Biosciences, 552845) were used to make the best fluorescence compensation settings for multicolor flow cytometric analysis.
7
Murine Lung Lavage for Cytokine Analysis
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Immediately after euthanasia, 1 ml aliquots of PBS were slowly infused in the murine lungs through the tracheostomy and then withdrawn gently. This lavage was repeated three times using the same syringe. The collected lavage uid was stored in a 10ml tube on ice. The uid was centrifuged at 1000rpm and 4°C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000g and 4°C for 10 min, stored at -80°C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.
8
Murine Lung Lavage and Cell Isolation
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Immediately after euthanasia, 1 ml aliquots of PBS were slowly infused in the murine lungs through the tracheostomy and then withdrawn gently. This lavage was repeated three times using the same syringe.
The collected lavage uid was stored in a 10ml tube on ice. The uid was centrifuged at 1000rpm and 4°C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000g and 4°C for 10 min, stored at -80°C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.
The collected lavage uid was stored in a 10ml tube on ice. The uid was centrifuged at 1000rpm and 4°C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000g and 4°C for 10 min, stored at -80°C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.
9
Immunophenotyping of Xenograft Samples
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The cells were washed with a staining solution (2% FBS in PBS) and resuspended in a staining solution containing fluorochrome-conjugated antibodies for 30 min. Propidium iodide (PI) was added to exclude dead cells from analysis. After staining, the cells were washed and analyzed using a Cytoflex flow cytometer (Beckman Coulter, Brea, CA, USA). Analysis was performed using CytExpert and FlowJo software. For xenograft sample analysis, BM cells were flushed from the femurs of mice and subjected to red blood cell lysis (Cat. R1010; Solarbio, Beijing, China) according to the manufacturer’s protocol. BM cells were then strained through a 70-µm mesh cell strainer and immunophenotyped using standard methodology.
10
Malignant Ascites Cell Processing
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The 10 cases of malignant ascites were centrifuged to collect the pelleted cells, which were then suspended in erythrocyte lysis buffer (Solarbio R1010) for 15 minutes to lyse the erythrocytes. Then the cell suspensions were pooled and pipetted into a new 50-ml conical tube through a 100 μm mesh, spun for 5 min at 500 g at 4°C and resuspended in 1×PBS to prepare for droplet-based scRNA-seq.
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