To determine the expression level of CXCR4 on lymphocytes, granulocytes, monocytes and T‐cell subsets, cynomolgus monkey blood was incubated with CD3‐PerCP (clone SP34‐2, BD552851), CD4‐FITC (clone L200, BD550628), CD8‐APC‐Cy7 (clone RPA‐T8, BD560662), CD95‐BV421 (clone DX2, BD562616), CD28‐BV510 (clone CD28.2, BD563075) per the manufacturers recommendations and 2.5 µg/ml MEDI3185‐AlexaFluor647 for 30 minutes at room temperature. Two milliliters of BD Pharm Lyse was added to lyse red blood cells, followed by washing with BD Cell Wash (BD Biosciences, San Jose, CA) and resuspended in 0.5 ml of BD Cell Wash. Samples were analyzed immediately on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA)
Cell wash
Manufactured by BD
Sourced in United States, Germany, Belgium, United Kingdom
The Cell Wash is a laboratory equipment used for washing and preparing cell samples. It is designed to separate cells from their surrounding media or solutions, ensuring the removal of unwanted components while preserving the integrity of the cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (9 mentions)
Pharm lyse, by BD (4 mentions)
Facscalibur, by BD (4 mentions)
Facsdiva software, by BD (3 mentions)
Cellquest pro, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Cellfix, by BD (3 mentions)
Live dead near ir stain, by Thermo Fisher Scientific (3 mentions)
Facscanto, by BD (3 mentions)
Guava easycyte system, by Merck Group (2 mentions)
Helix np nir, by BioLegend (2 mentions)
Facs lysing solution, by BD (2 mentions)
Facsfix, by BD (2 mentions)
Facs lyse, by BD (2 mentions)
Matched isotype control, by BD (2 mentions)
Novoexpress, by Agilent Technologies (2 mentions)
Facsaria 3, by BD (2 mentions)
Chrompure mouse igg, by Jackson ImmunoResearch (2 mentions)
Cd45 fitc, by BD (2 mentions)
Stain buffer, by BD (2 mentions)
41 protocols using cell wash
1
Cytometric Analysis of CXCR4 Expression
2
Measurement of Cellular Oxidative Stress
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ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Cells were preincubated for 20 minutes at 37 °C. The indicated agonists and DHR (2 µM) were then added for 15 minutes. To stop the reaction, cells were placed on ice, washed, and resuspended in Cell Wash (BD Bioscience) containing 0.5% PFA and 50 nM of the cell-impermeant far-red emitting nucleic acid stain Helix NP NIR (Biolegend) to gate for live cells. 10,000 live cells per condition were analysed on a Guava easyCyte™ System (Millipore, Burlington, MA, USA) for median fluorescence intensity. Agonist-mediated ROS production was expressed as percentage of the signal detected in untreated cells. The FPR2/ALX specific agonist WKYMVm was used as a positive control.
3
Characterization of T Cell Activation
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The expression of surface activation markers was studied on fresh blood kept at room temperature until it is used. CD38 and HLA-DR expression was measured on CD4 and CD8 T cells by six-color flow cytometry using a whole blood cell procedure. Briefly, 50 μL of whole blood were incubated with a mixture of antibodies (all from BD Biosciences, San Jose, CA, USA) namely CD3 − fluorescein isothiocyanate (FITC), CD8 −peridinin-chrorophyll-protein-cyanin 5.5 (PerCP-Cy5.5), CD4 − phycoerythrin cyanin 7 (PC7), CD45 − allophycocyanin 7 (APC-Cy7) and either CD38 − phycoerythrin (PE) and HLA-DR − allophycocyanin (APC) or their irrelevant counterparts, for 15 min in the dark. The red blood cells were lysed using 2 ml of FACS lysing solution (BD Biosciences), washed before being suspended in 500 μL of Cell Wash (BD Biosciences). A minimum of 10,000 lymphocytes was acquired for each condition. Flow cytometric acquisition and analysis were performed on a FACSCanto flow cytometer and analysis was performed using FACSDiva software (v6.1, BD Biosciences).
4
Quantitative Analysis of CD127 Expression
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Five microliters of anti-human CD127 PE (IL7Rα) (BD Biosciences) or matched isotype control (BD Biosciences) was added to 100 μl of blood or 106 PBMCs, incubated for 10 min at room temperature. Cells were lysed with FACsLyse (BD Biosciences) for 10 min, and the remaining cells were washed with Cell Wash (BD Biosciences) before being fixed (FACsFix, BD Bioscience). Ten thousand lymphocytes were acquired (FACsCalibur, BD Biosciences) and analyzed (Cell Quest Pro, BD Biosciences).
5
Multiparametric Flow Cytometry Analysis
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For surface staining, cells were incubated with diverse combinations of fluorochrome-labeled antibodies against CD11c (clone N418), MHC class II (clone M5/114.15.2), CD4 (clone RM4-5), Ly6C (clone AL-21), CD103 (clone 2E7), CD11b (clone M1/70), Bst2 (clone eBio927), B220 (clone RA3-6B2), SIRP-α (clone P84), CCR9 (clone eBioCW1.2), or Siglec-H (clone eBio440c) for 15 min in the dark at 4°C as indicated. Finally, the cells were washed with Cell Wash (BD Biosciences). Appropriate isotype controls were used to define the threshold of negative versus positive staining. All antibodies were purchased from BD Biosciences or from eBioscience (Frankfurt am Main, Germany). Data were acquired using a FACSCalibur or FACSCanto II (BD Biosciences). Data analysis was performed using CellQuest Pro, FACSDiva Software (both BD Biosciences), or NovoExpress (ACEA Biosciences, San Diego, CA, USA).
6
Flow Cytometric Analysis of TMIGD1
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MDCK II Tet-Off cell lines stably expressing Flag-TMIGD1 constructs were washed in PBS, incubated for 10 min at 37 °C in PBS / 5 mM EDTA followed by incubation with Accutase® (Sigma Aldrich #A6964) for 10 min at 37 °C. For the analysis of cell surface-localized proteins, cells were incubated with rabbit pAb anti-Flag in CellWASH (BD #349524) containing 5% BSA (1 h, 4 °C), followed by incubation with AlexaF488-conjugated anti-rabbit pAb (45 min, 4 °C). For intracellular stainings, cells were washed, fixed in PBS / 2% PFA, and incubated with primary and secondary antibodies in the presence of 0.5% saponin (Sigma Aldrich #A47036). After washing, cells were resuspended in CellWASH containing 5% BSA at 106 cells per ml and analyzed by flow cytometry using a Guava® easyCyte™ flow cytometer (MerckMillipore). For each sample, 10.000 cells were measured. Results were analysed using the Guava® InCyte™ software (MerckMillipore) and are plotted as histograms.
7
Flow Sorting and NGS Clonality Analysis
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Diagnostic samples were processed for cell sorting. Target populations were purified using a BD FACS Aria III flow sorter (Becton Dickinson [BD], San Jose, CA, USA) as described previously [68 (link)]. A nonfixative lysis agent (PharmLyse, BD) was used, and the cells were diluted in phosphate-buffered saline (Cell Wash, BD). The cells were sorted in ‘Purity’ mode and collected in Eppendorf tubes containing relevant buffer. A total of 3 to 5 million cells were sorted in duplicate for clonality using NGS.
8
Measuring Sperm Mitochondrial Membrane Potential
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Sperm mitochondrial membrane potential (MMP) was measured using the reagent 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidaz-olylcarbocyanine iodide, (JC-1; Thermo Fisher Scientific,
Waltham, MA, USA). Approximately 1000 µl CellWASH™ (BD Biosciences), containing one million spermatozoa, and 0.5 µl of 3 mM JC-1 were added and the samples were incubated at
37oC for 40 min in the dark. The stained samples were analyzed using a BD LSR flow cytometer (BD Biosciences). Excitation of JC-1 in stained cells was obtained by an argon-ion
laser (488 nm). Emitted fluorescence was detected using bandpass filters 530/28 nm (FL1, green fluorescence) and 575/26 nm (FL2, orange fluorescence). A total of 30,000 events was evaluated
and classified in 2 distinct groups: spermatozoa with high respiratory activity MMP-H (orange fluorescence) and those with low respiratory activity MMP-L (green fluorescence).
Waltham, MA, USA). Approximately 1000 µl CellWASH™ (BD Biosciences), containing one million spermatozoa, and 0.5 µl of 3 mM JC-1 were added and the samples were incubated at
37oC for 40 min in the dark. The stained samples were analyzed using a BD LSR flow cytometer (BD Biosciences). Excitation of JC-1 in stained cells was obtained by an argon-ion
laser (488 nm). Emitted fluorescence was detected using bandpass filters 530/28 nm (FL1, green fluorescence) and 575/26 nm (FL2, orange fluorescence). A total of 30,000 events was evaluated
and classified in 2 distinct groups: spermatozoa with high respiratory activity MMP-H (orange fluorescence) and those with low respiratory activity MMP-L (green fluorescence).
9
Lymphocyte Activation Assay with PHA
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Heparinized whole venous blood, 2 mL was collected (LH 68 IU BD-Plymouth, United Kingdom, 5 mL) from each subject and was separated equally into two tubes - control tube and a PHA-M stimulated sample. To the stimulated test tube 20 μg/mL PHA-M (Roche Diagnostics GmbH, Germany) was added and the two samples were incubated for 4 h at 37 °C, 5% CO2. The samples were gently shacked, at regular intervals, on a multispeed vortex (MSV-3500 BioSan LV). Afterwards, 100 μL blood from each tube was labeled with monoclonal anti-CD3 FITC (for determination of the T lymphocytes) and anti-CD69 PE, an early activation marker for T cells (BD Biosciences, United States) and incubated for 30 min, at room temperature (RT) in the dark. Followed a lysis of erythrocytes (BD FACS Lysing Solution, BD Biosciences, United States), then centrifuging at 1300 rpm for 10 min and double washing (CellWash, BD Biosciences, United States). Subsequently, cells were fixed with 200 μL CellFIX (BD Biosciences, United States) and were analyzed with BD FACSCalibur flowcytometer using the Cell Quest software for data acquisition and analysis. Then 20000 lymphocytes were counted and analyzed for expression of CD69. The results obtained for each patient and healthy subject were analyzed for PHA-stimulated and unstimulated lymphocytes.
10
FACS Staining of PMN Activation Markers
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Analyses of the expression of cell surface activation markers were performed using differential FACS staining. PMN were labeled with CD66b-FITC (clone 80H3, AbD Serotec, Puchheim, Germany), CD62L-APC (clone DREG-56, BioLegend, Fell, Germany), CD63-V450 (clone H5C6, BD Biosciences, Heidelberg, Germany), and CD69-PE (clone L78, BD Biosciences, Heidelberg, Germany) at 4°C for 30 min, washed with CellWash (BD Biosciences, Heidelberg, Germany), and centrifuged at 300 g for 5 min. Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences, Heidelberg, Germany). The software FlowJo 7.6.4 (BD Biosciences, Heidelberg, Germany) was used for analysis.
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