BD flow cytometry tubes were labeled, and negative control tubes and experimental tubes were prepared for fluorescent antibody staining. FITC CD45 5 μL (BD Biosciences) and APC CD41a (BD Biosciences) were added to the experimental tubes. An appropriate volume of isotype control was added to the negative control tubes. Then, 50 μL of unstimulated or activated fresh whole blood was added within 10 minutes of blood collection, with an appropriate amount of PBS to make up the final volume of 100 μL. The tubes were incubated for 20 minutes at room temperature in the dark. Then, 1 ml of lysing solution (Becton, Dickinson, and Co.) was added to each tube and vortexed for 10 minutes and centrifuged at 1300 rpm for 5 minutes. The supernatant was discarded, and 500 μL of 1% paraformaldehyde was added. Then, the samples were subjected to flow cytometry (BD FACS Caliber, BD, USA).
Lysing solution
Manufactured by BD
Sourced in United States, Germany
Lysing solution is a laboratory reagent used to disrupt the cell membrane and release the cellular contents, including nucleic acids, proteins, and other biomolecules. It is a core component in various molecular biology and biochemical applications, such as DNA/RNA extraction and cell lysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (16 mentions)
Facscalibur, by BD (13 mentions)
Permeabilizing solution 2, by BD (7 mentions)
Facscanto 2 flow cytometer, by BD (6 mentions)
Facsdiva software, by BD (5 mentions)
Spectroflo software v3, by Cytek (4 mentions)
Brilliant stain buffer plus, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Facscanto, by BD (3 mentions)
Facscan flow cytometer, by BD (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (3 mentions)
Aurora cytometer, by FlowJo (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Canto 2, by BD (3 mentions)
Trucount tube, by BD (3 mentions)
Percp cy5, by Thermo Fisher Scientific (2 mentions)
Facs permeabilizing solution, by BD (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
136 protocols using lysing solution
1
Quantification of Platelet-Leukocyte Interactions
2
Multicolor Flow Cytometry Analysis
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Flow cytometry was used to determine the percentage of cells that were positively labeled with antibodies [phycoerythrin (PE), fluorescein isothiocyanate (FITC), and allophycocyanine (APC) (eBioscience and ABCAM)] in different organs and BALF from the animals. The following staining parameters were employed: eosinophils were identified by SIGLEC-f (clone ESO-2440) and CD11b (clone M1/70), T cells were identified by CD3 (clone 17A2), CD45 (clone 30-F17), CD4 (clone RM4-4), CD8 (clone 53-6.7), TCR (clone UC7-13D5), B cell CD19 (Ebio1D3), and CD22 (clone ab25369), and NK cells (clone PK136). The isotype controls were mouse immunoglobulin IgG2b l FITC, rat IgG2a k PE, and mouse IgG1 k APC. Cells (106 cells/mL) were labeled with 1 mg of monoclonal antibody and incubated for 20 min at room temperature (RT), and erythrocytes were lysed by adding 2 mL of 10% lysis solution (Lysing Solution, Becton Dickinson). Next, tubes were centrifuged at 1,500 rpm for 10 min, the supernatant was discarded, and the cell pellet was washed twice with PBS. After incubation, the cells were resuspended in 1% PFA/PBS and analyzed using a FACSCanto flow cytometer, and the data were analyzed with FlowJo software (Tree Star, Inc., USA). Positive controls and FMO were performed for each tissue evaluated.
3
Flow Cytometry-Based Lymphocyte Isolation
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Peripheral blood samples, collected using ethylene diamine tetra acetic acid (EDTA) as an anticoagulant, were stained for flow cytometry purposes within biosafety level 2 laboratories. Each sample underwent a fixation/erythrocyte lysis step using Lysing solution (Becton Dickinson, BD, Biosciences, La Jolla, CA, USA) under gentle agitation (15 min, room temperature). Samples were then stained by adding the reagent mix reported in Table S1 (30 min, 4 °C), washed, and postfixed (BD Biosciences, La Jolla, CA, USA); then, CD3+ T lymphocytes and CD19+ B cells were separated via fluorescence-activated cell sorting (FACSAria III, BD Biosciences, San Jose, CA, USA) using a 100 μm nozzle [176 (link)]. A high level of purity for each isolated population was achieved (>90%).
4
Leukocyte Phenotyping by Flow Cytometry
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Leukocyte phenotyping was conducted by dual-colour flow cytometry using a whole blood lysis technique and monoclonal antibodies (using phycoerythrin-conjugated CD14, fluorescein isothiocyanate-coupled CD45, and fluorescein isothiocyanate-coupled HLA-DR (all from Becton Dickinson, Heidelberg, Germany)). For technical details, see [27 (link)]. In short, diluted heparinized blood containing 5000–10,000 leukocytes/μL was added to 20 μL of antibody pairs for a final volume of 300 μL and incubated in the dark for 15 min. Erythrocytes were lysed with lysing solution (Becton Dickinson, Heidelberg, Germany) and washed once with phosphate-buffered saline. Measurement of stained cells was performed on a FACSCanto (Becton Dickinson, Heidelberg, Germany). Monocytes were defined using scatter characteristics and CD14/CD45 staining. The monocyte population was analysed for HLA-DR expression which was expressed as mean fluorescence intensity (MFI).
5
Quantifying Endothelial Progenitor Cells Post-Myocardial Infarction
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The number of circulating CD34+/CXCR4+ cells, an endothelial progenitor cell (EPC) in the peripheral blood was measured as CD34+ and CXCR4+ double positive cells on the day 7 after MI in each group. Briefly, a 100 μL aliquot of heparinized whole blood was subjected to incubation with fluorescein isothiocyanate (FITC)‐conjugated mouse anti‐human CD34 (Bio Rad Laboratories Inc, Hercules, CA) and Phycoerythrin (PE)‐ conjugated mouse monoclonal anti‐human CXCR‐4 (R&D System, Inc.) at 4°C for 30 minutes. RBCs were lysed by adding Lysing Solution (Becton Dickinson, Franklin Lakes, NJ). After the cells were washed with phosphate buffered saline (PBS), they were analyzed by S3TM Cell Sorter (488/561 nm) 145‐1002 (Bio‐Rad Laboratories, Inc.) using S3 ProSort Soft Ware.
6
Confirming Hematopoietic System Reconstitution
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Peripheral blood was analyzed for GFP expression using fluorescence-activated cell sorting (FACS) to confirm the re-established haematopoietic system by transplanted male GFP BM. Terminal blood samples (0.7 ml) were taken by cardiac puncture into heparin-coated tubes. A volume of 0.2 ml of blood was aliquoted into separate 15 ml Falcon tubes. Next, 1.8 ml of lysing solution (Becton-Dickinson, Bedford, USA) was added to each tube, vortexed and left to incubate for 3 minutes in the dark. After incubation, 13 ml of PBS were added to each tube to dilute the lysing solution and centrifuged at 400 × g for 4 minutes. The resultant supernatant was removed and the pellet was resuspended in cold PBS on ice. The cells were analyzed by a FACS Aria (Becton Dickinson Biosciences) following the instrument configuration and sorting procedure recommended by the manufacturer. Each analysis included at least 10 000 events.
7
Mouse Lymphocyte Subset Identification
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Anti-mouse CD4 and CD8 mAbs, labeled with peridinin-chlorophyll-a-protein-cyanine 5.5 complex (PerCP-Cy5.5) and phycoerythrin (PE), were purchased from eBioscience (Thermo Fisher, Waltham, MA, USA). Fifty μL of venous mouse blood were mixed with 50 μL of normal saline and incubated in the dark with 1 μL of PerCP-Cy5.5 labeled anti-CD4 and 1 μL of PE-conjugated anti-CD8a for 30 min. One ml (5% v/v) of lysing solution (Becton Dickinson, Franklin Lakes, NJ, USA) was then added for 5 min to ensure the complete lysis of the erythrocytes, and the surviving cells were washed twice with PBS, with centrifugation at 500 g for 5 min between each step. Finally, the cells were resuspended in 150 μL of PBS and analyzed in a FACScan flow cytometer (Becton Dickinson).
8
Peripheral Blood Lymphocyte Immunophenotyping
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Anti-Mouse CD8a and Anti-Mouse CD4, labelled with phycoerythrin (PE) and PerCP-Cy5.5, respectively, were purchased from eBioscience (San Diego, CA, USA) Each test contained 50 µL of peripheral blood sample, and then incubated 0.25 µL PE labeled Anti-Mouse CD8a and 0.25 µL PerCP-Cy5.5 labeled Anti-Mouse CD4. After incubating for 20 min in the dark, 2 mL lysing solution (Becton Dickinson, Franklin Lakes, NJ, USA, 10%, v/v) was added to make the lysis of erythrocytes more complete and surviving cells were washed twice with PBS, with centrifugation between each step for 5 min at 500 g. FACScan flow cytometer was used for FCM analysis (BD Biosciences, San Jose, CA, USA). First, an electronic gate was set on lymphocytes and then gated CD4+ T cells in Y axis and CD8+ cells as figure. Dead cells were excluded according to the forward scatter and side scatter.
9
Quantification of CD4+ and CD8+ T Cells
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Flow cytometry was used to assay CD4+ and CD8+ lymphocyte populations in peripheral blood. The fluorescein isothiocyanate (FITC)-CD4+ and phycoerythrin (PE)-CD8+ mouse anti-chicken antibodies (SouthernBiotech, Birmingham, USA) and blood samples were added to each tube and mixed by gentle shaking at 4 °C for 30 min. Erythrocytes were then lysed with 2 mL of Lysing Solution (Becton Dickinson (BD), Franklin Lakes, NJ, USA) at room temperature for 20 min. The lysate was centrifuged at 1500×g for 5 min and the supernatant was removed. The pellet was resuspended in phosphate-buffered saline (PBS). The lysate was centrifuged again at 1500×g for 5 min, the supernatant was removed, and the pellet was resuspended in 500 mL of PBS for assay performed on a flow cytometer (Coulter XL FCM, Beckman Coulter, Brea, CA, USA).
10
Flow Cytometric Immunophenotyping of CD4 and CD8
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Mouse anti-mouse CD4 and CD8a (Lγ-2) mAbs, labeled with fluorescein isothiocyanate (FITC) and phycoerythrin (PE) respectively, were purchased from eBioscience (USA). 20 μl venous bloods were mixed with 30 μl normal saline, and incubated with 0.4 μl FITC-conjugated anti-CD4, 0.4 μl PE labeled anti-CD8a and 2.2 µl PBS for 20 min in the dark. 2 ml (10% v/v) lysing solution (Becton Dickinson, USA) was then added for 5–10 min to ensure complete lysis of erythrocytes, and surviving cells were washed twice with PBS, with centrifugation between each step for 5 min at 500 g. Finally, the cells were resuspended in 100 μl PBS and analyzed in a FACScan flow cytometer (Becton Dickinson).
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