Flow cytometry to detect cell apoptosis: Spread 2.5×106 cells/well in a 6-well plate, and after they adhere to the wall, treat them with chloroquine for 12-24 hours. Collect the cell pellets when the cell status changes under the microscope, and use the AnnexinV, FITC Apoptosis Detection Kit (Dojindo, Japan) for detection. Flow cytometry to detect immune cells: After the treatment, the mouse spleen cells were separated and red blood cell lysate (Beyotime Biotechnology, Shanghai, China) was added. Resuspend the cells in PBS and count them. Take about 2×106 cells from each experimental group, resuspend them in 200 μl of PBS, and add the corresponding fluorochrome-labeled antibodies (CD3/FITC, CD4/PE and CD8/APC) (all purchased from BioLegend, USA). Incubate for 30 minutes at 4°C in the dark. Flow cytometry analysis of fluorescence intensity.
Red blood cell lysate
Manufactured by Beyotime
Sourced in China
Red blood cell lysate is a reagent used to extract cellular contents from red blood cells. It disrupts the cell membrane to release the intracellular components, including proteins, nucleic acids, and other biomolecules. This product is intended for use in various research and analytical applications that require access to the contents of red blood cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BioLegend (2 mentions)
Cd11b pe, by BioLegend (2 mentions)
Cd44 apc, by BioLegend (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Cd8 apc, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Beyotime (1 mentions)
Trypsinization, by Beyotime (1 mentions)
6 protocols using red blood cell lysate
1
Apoptosis and Immune Cell Analysis
2
Isolation and Culture of CD44+/CD45-/CD11b- Cells
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Adult male C57BL/6 mice were sacrificed by cervical dislocation and then sterilized and immersed in 75% ethanol for 5–10 min. The femur and tibia were separated under aseptic conditions and cut into pieces with a volume less than 3 mm3. Red blood cells were lysed with red blood cell lysate (C3702-120 mL, Beyotime), and after centrifugation, the fragments were resuspended in culture medium, inoculated in a culture flask, and placed in an incubator at 37 °C, 5% CO2 and 95% O2 with saturated humidity. Cells were passaged each time the cells formed a certain number of colonies in the culture flask. Third-generation cells incubated with APC-CD44 (103012, Biolegend), FITC-CD45 (103107, Biolegend) and PE-CD11b (101207, Biolegend) were used for flow sorting, and the CD44+/CD45−/CD11b− cells were sorted by a Beckman flow cytometry sorting system (Moflo-XDP, Backman). The sorted cells were collected in a sterile flow tube, transferred to a cell culture flask to continue culturing, and the culture medium was replaced every 48 h.
3
Enzymatic Dissociation of Tumor Tissue
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Tumor tissue was minced into small pieces of 1-3 mm3 and then incubated in the complete medium that contained type I collagenase (final concentration 1 mg/ml) (Sigma, Cat#C0130) and DNase I (final concentration 100 U/ml) (Acmec, Cat#D61780) at 37°C with constant shaking (100 rpm) for 4 hours. The tissue suspension was washed three times with PBS at 1000 rpm for 5 minutes each after passing through a 70 μm cell strainer. Cells were lysed using a red blood cell lysate (Beyotime, Cat#C3702) and cells were allowed to stand on ice for 5 minutes. The single-cell suspension was obtained after three washes using PBS at 1000 rpm for 5 minutes.
4
SARS-CoV-2 S2 Peptide ELISpot Assay
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IFN-γ ELISpot assays were conducted as previously described [26 (link)]. Briefly, ELISpot plates were blocked using 1640 containing 10% FBS at 37oC for 2 h. Spleen cell suspension was prepared, and red blood cells were lysed by red blood cell lysate (Beyotime Biotechnology). Cells were counted and added into the plates (1 × 106 per well) together with 1 µg/mL SARS-CoV-2 S2 full-length scan peptide library (synthesized by GL Biochem) and incubated at 37oC for 48 h. Plates were washed 5 times with sterile PBS before incubation with 1 µg/mL biotin-anti-IFN-γ antibody for 2 h at room temperature. After washing with PBS, streptavidin-ALP at 100 µg/well was added and incubated for 1 h at room temperature. Substrate solution (BCIP/NBT) was added to visualize the enzyme-linked reaction, and the antigen-specific spots were counted using an AID ELISpot Reader System (AID, Strasburg, Germany).
5
Isolation and Culture of Peritoneal Macrophages
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After two lavages, the peritoneal lavage fluid was collected and centrifuged by density centrifugation at 4°C and 2000g for 15 min, and the supernatant was discarded. If there were numerous red blood cells after centrifugation, two to three times the volume of red blood cell lysate (Beyotime) was added, mixed, and centrifuged again after resting for two minutes. After discarding the supernatant, DMEM complete culture medium (Beyotime) including 10% fetal bovine serum (Beyotime) and 1% penicillin-streptomycin (Beyotime) was added, resuspended, and inoculated into 12-well plates for culture for 4 h. The medium was changed after the macrophages adhered, and the macrophages were collected by trypsinization (Beyotime) after culturing for 24 h.
6
Isolation and Characterization of Murine Mesenchymal Stem Cells
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The adult male C57BL/6 mice were sacri ced by cervical dislocation and then sterilized and immersed in 75% ethanol for 5-10 minutes. The femur and tibia were separated under aseptic conditions and cut into pieces with a volume less than 3mm 3 . Red blood cells were lysed with red blood cell lysate (C3702-120mL, Beyotime), and after centrifugation, the fragments were re-suspended in culture medium and inoculated in a culture ask, and placed in an incubator at 37°C, 5% CO 2 and 95% O 2 with saturated humidity. Cells were passaged each time when the cells form a certain number of colonies in the culture ask. The third-generation cells which were incubated with APC-CD44 (103012, Biolegend), FITC-CD45 (103107, Biolegend) and PE-CD11b (101207, Biolegend) were used for ow sorting, and the cells of CD44 + /CD45 -/CD11b -were sorted by Beckman ow cytometry sorting system (Mo o-XDP, Backman). The sorted cells were collected in a sterile ow tube, transferred to a cell culture ask to continue culturing, and replaced the culture medium every 48h.
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