Lymph nodes were transferred to a meshed-cap vial (35 µm mesh), pressed through it with 400 µL of PBS and counted using an automated cell counter (Sysmex, Norderstedt, Germany). Cells were resuspended in FACS buffer (FBS 2%, EDTA 2 mM in PBS) and stained with eBioscience Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific), followed by incubation with Fc-gamma receptor block (Becton Dickinson). The cells were then stained with fluorochrome-conjugated antibodies: CD3-BV510, CD4-A488 or CD4-APC, CD8-FITC or CD8-BV421 (BioLegend, San Diego, CA, USA). The cells were immediately acquired using FACSVerse (Becton Dickinson) and the data were analyzed with FlowJo software Version 10 (FlowJo_v10.6.1, Ashland, OR, USA). Forward and side scatter gates were used to discriminate doublets and debris (FSC-A, FSC-H, SSC-A × SSC-H). Fluorescence minus one was used as control. Only viable cells were included in the analysis.
Automated cell counter
Manufactured by Sysmex
Sourced in Germany, Japan
The Automated Cell Counter is a compact, fully automated device that accurately counts and analyzes cells in a variety of biological samples. It provides precise cell counts and viability assessments to support research and diagnostic needs.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 gk1, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti mouse cd16 cd32, by BD (2 mentions)
Ebioscience fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Fc gamma receptor block, by BD (1 mentions)
Cd8 bv421, by BioLegend (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Cd8 fitc, by BioLegend (1 mentions)
Cd3 bv510, by BioLegend (1 mentions)
Facsverse, by BD (1 mentions)
Spss for windows v 16, by IBM (1 mentions)
Cd25 pc61, by BD (1 mentions)
Cd44 im7, by BioLegend (1 mentions)
Rm4 5, by BD (1 mentions)
Cd24 m1 69, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
40 μm cell strainer, by Thermo Fisher Scientific (1 mentions)
Cd8a 53 6, by BioLegend (1 mentions)
Cd19 apc cy7 a, by BD (1 mentions)
16 protocols using automated cell counter
1
Lymph Node Cell Counting and Phenotyping
2
Evaluation of β-Thalassemia Heterozygosity
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Complete blood counts were performed on 486 unrelated β-thalassaemia heterozygous patients that were diagnosed in our centre using an automated cell counter (Sysmex, Tokyo, Japan). Histograms and tables of descriptive statistics of Hb, MCV, MCH, HbA and HbA2 were generated and compared by gender (n>15). Also, HbA, HbA2, MCV, MCH and Hb were compared among different genotypes of β-thalassaemia (n>10). Continuous variables were compared using analysis of variance. For multiple comparisons, a post hoc analysis was used when appropriate. Duncan's multiple range test was used to decrease type I error rates. All reported p-values are two-sided and were statistically significant if p<0.05. All statistics in this study were computed with SPSS V.16 for Windows.
3
Tail Bleeding Assay in Mice
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Litter-matched control and mutant mice were anesthetized without reawakening by intraperitoneal injection of fentanyl (0.05 mg kg−1), midazolam (5 mg kg−1) and medetomidin (0.5 mg kg−1). The tail tip (5 mm) was cut off with a scalpel. Blood drops were collected every 20 s on a filter paper without touching the lesion until blood flow stopped. Bleeding exceeding 15 min was stopped using tissue glue. After completion of the tail bleeding assay, blood was drawn from the retro-orbital plexus of each anesthetized animal and platelet counts were determined using an automated cell counter (Sysmex GmbH). Only mice with a normal range of platelet counts (800–1300 × 106 mL−1) were included for analysis of the bleeding time.
4
Immunophenotyping of Lymphoid Cells
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Single cells from thymus, spleen, and lymph nodes were prepared by passing the tissue through a 40 μm cell strainer (Thermo Fisher) using PBS and a syringe plunger. Erythrocytes in blood and spleen were lyzed in lysis buffer (0.16 M NH4Cl, 0.13 M EDTA, and 12 mM NaHCO3), the cells were washed in flow cytometry buffer (2% fetal bovine serum and 2 mM EDTA in PBS) and counted in an automated cell counter (Sysmex). After FcR-blockage (anti-mouse CD16/CD32, BD Biosciences), antibodies specific for the following markers were used: CD4 (GK1.5, Biolegend or RM4-5, BD Biosciences), CD8a (53-6.7, Biolegend), CD44 (IM7, Biolegend), CD25 (PC61, BD Biosciences), CD24 (M1/69, BD Biosciences), and Qa-2 (1-1-2, BD Biosciences). Immunostained cells were analyzed on a FACS Canto II, Accuri C6, or FACS Aria (BD Biosciences). Data were analyzed using FlowJo (Tree Star) and fluorochrome-minus-one staining was used as controls.
5
Granulocyte Isolation and Characterization
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First, granulocyte purity after each method was assessed through cell counting using an automated cell counter (Sysmex, Brasil), and the cells from the highest purity methods were characterized by flow cytometry (LSRFortessa, Bdsciencis). Isolated granulocytes were phenotyped by negative markers (CD14, CD19 and CD3) and positive markers (CD45, CD15 and CD13).
For this purpose, 106 isolated cells in 100 µL were used, and the following granulocyte-specific antibodies were used to confirm their identity as white cells and neutrophils: 5 µL of CD3-AlexaFluor 700-A; 2 µL of CD14-PE-Cy7-A; 5 µL of CD19-APC-Cy7-A (BD Biosciences, EUA) to exclude monocytes and dendritic cells, lymphocytes and lymphocytes B, respectively; 8 µL of CD45-PercP-Cy5.5-A; 5 µL CD15-FITC-A and 5 µL of CD13-PE-A (BD Biosciences, EUA).
For this purpose, 106 isolated cells in 100 µL were used, and the following granulocyte-specific antibodies were used to confirm their identity as white cells and neutrophils: 5 µL of CD3-AlexaFluor 700-A; 2 µL of CD14-PE-Cy7-A; 5 µL of CD19-APC-Cy7-A (BD Biosciences, EUA) to exclude monocytes and dendritic cells, lymphocytes and lymphocytes B, respectively; 8 µL of CD45-PercP-Cy5.5-A; 5 µL CD15-FITC-A and 5 µL of CD13-PE-A (BD Biosciences, EUA).
6
Phenotyping Immune Cell Subsets in Murine Bone Marrow and Thymus
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Bone marrow (BM) cells were obtained by flushing femurs and/or humerus with PBS, and erythrocytes were lysed. Thymi were pressed through 70 µm cell strainers to obtain single-cell suspensions. Cellularity was determined using an automated cell counter (Sysmex, Noderstedt, Germany). Purified rat anti-mouse CD16/32 (BD Biosciences, Franklin Lakes, NJ, USA) was added to block antibodies from binding to Fc receptors, prior staining of surface markers. Cells were stained with the following fluorochrome-conjugated antibodies; antimouse CD8a PE (clone 53-6.7, Biolegend, San Diego, CA, USA), anti-mouse CD4 BD Horizon V450 (clone RM4-5, BD), anti-mouse CD117/ckit APC (clone 2B8, Biolegend), anti-mouse CD45R/B220 PerCP (clone RA3-6B2, Biolegend), anti-mouse CD19 BD Horizon V450 (clone 1D3, BD), anti-mouse IgM PE (clone 1B4B1, Southern Biotechnology, Birmingham, AL, USA) and anti-mouse CD93 PE-Cy7 (clone AA4.1, Biolegend). Fluorescenceminus-one (FMO)-stained samples were used as controls. Data were acquired on a BD FACS Canto II and analyzed using Flow Jo 8.8.6 (Three Star, Ashland, USA).
7
Evaluating T-cell Activation and Proliferation
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Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood of healthy volunteers after informed written consent using standard density gradient centrifugation (Lymphoprep). PBMCs (1.25 × 105/well) were cultured in a 48-well plate in the presence of 0.5 μg/mL agonistic CD3 mAb (UCHT-1) and indicated concentrations anti-PD-L1:TRAIL, anti-EpCAM:TRAIL, or PD-L1 mAb. After 72 h, total cell number was quantified using an automated cell counter (Sysmex) and culture supernatants were stored at −20°C. IFNγ levels in culture supernatant were determined by IFNγ ELISA (eBioscience). Where indicated, freshly isolated PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) (CellTrace CFSE Cell Proliferation Kit, Invitrogen), and after 72 h of respective treatment the cell proliferation was analyzed by flow cytometry within the live PBMCs (defined by FSC/SSC gating).
For CMV specific responses, freshly isolated PBMCs from CMV negative and positive donors were cultured in 96-well plates (1.5 × 105/well) in the presence of CMV pp65 according to manufacturer's instructions (Miltenyi Biotec). After 96 h, culture supernatants were stored at −20°C and secreted IFNγ was determined by IFNγ ELISA.
For CMV specific responses, freshly isolated PBMCs from CMV negative and positive donors were cultured in 96-well plates (1.5 × 105/well) in the presence of CMV pp65 according to manufacturer's instructions (Miltenyi Biotec). After 96 h, culture supernatants were stored at −20°C and secreted IFNγ was determined by IFNγ ELISA.
8
Preparation of Platelet-Rich and Platelet-Free Plasma
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Venous blood from healthy consenting volunteers not taking any medication was obtained from the biobank of the Leiden University Medical Center. All healthy volunteers signed a written informed consent. The Medical Ethics Committee of the Leiden University Medical Center approved the study protocol.
Blood was collected into Vacutainer® tubes containing ACD (Becton Dickinson). Whole blood was centrifuged at 200 g for 20 min at room temperature to prepare platelet‐rich plasma (PRP). PFP was prepared by centrifuging PRP twice at 1200 g for 15 min at room temperature and the depletion of platelets was confirmed by automated cell counter (Sysmex). All functional assays were performed in freshly prepared PRP and PFP.
Additional methods are available in the supporting information AppendixS1 .
Blood was collected into Vacutainer® tubes containing ACD (Becton Dickinson). Whole blood was centrifuged at 200 g for 20 min at room temperature to prepare platelet‐rich plasma (PRP). PFP was prepared by centrifuging PRP twice at 1200 g for 15 min at room temperature and the depletion of platelets was confirmed by automated cell counter (Sysmex). All functional assays were performed in freshly prepared PRP and PFP.
Additional methods are available in the supporting information Appendix
9
Copy Number Variation Analysis in β-Globin Gene Cluster
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The copy number variation in the β‐globin gene cluster (NC_000011.10) was performed using multiplex ligation‐dependent probe amplification (MLPA) following the manufacturer's instructions (MRC Holland, Amsterdam, the Netherlands). Two commonest deletional HPFH, Chinese Gγ(Aγδβ)0 thalassemia and Southeast Asia HPFH (SEA‐HPFH) deletion were identified by Gap‐PCR (He et al., 2018 ). One intractable case was further sequenced using targeted next‐generation sequencing following methods from a previous report (Shang et al., 2017 ). Complete blood counts were performed using an automated cell counter (Sysmex, Tokyo, Japan), and whether individuals carried 17 common β‐globin gene mutations were tested using previously described methods (Zhang et al., 2012 ).
10
Isolation and Apoptosis Induction of PBMCs
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Human peripheral blood mononuclear cells (PBMC) were isolated from four healthy male volunteers by venous blood draw and density gradient centrifugation with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Sweden). PBMCs (25 × 106 cells/ml) were resuspended in serum-free medium (CellGro, CellGenix, Freiburg, Germany). An automated cell counter (Sysmex Inc., USA) was used to determine cell count. PBMCs were gamma-irradiated with 60 Gy to induce apoptosis. Induction of apoptosis was confirmed by annexin V-fluorescein/propidium iodide (FITC/PI) co-staining (Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometer. At 20h after irradiation 58% of PBMCs were annexin V/PI positive (supplementary Fig. S1 ). CM was collected from cultures at 2, 4, and 20 h time points, and then, centrifuged (500 × g for 9 min) to remove cell debris. CM was stored at −80 °C for subsequent protein and lipid analyses. Fresh CM was used for microparticle and exosome separations.
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