Since scRNAseq needs to be performed on fresh tissue, snRNAseq was chosen instead given its ability to be performed on previously frozen samples. snRNAseq samples were stored in liquid nitrogen for less than 5 months before processing. Nuclei were isolated using the Chromium Nuclei Isolation Kit (10X Genomics). snRNAseq was performed using the 10× Genomics Chromium X platform with 10× Genomics 3′ gene expression kit. Cell count and viability of the nuclei were verified on the ThermoFisher Countess 3 cell counter and EVO imager microscope. Single nuclei were parsed and proceeded into library preparation following manufacturer protocol. Quality control of the libraries was performed on the Agilent BioAnalyzer and Kapa library quantification. Samples were sequenced on the Illumina Novaseq6000 SP and data was packaged using the 10× Genomics CellRanger pipeline. The snRNAseq and spatial transcriptomic samples were patient-matched to increase the reliability of results during single-cell mapping and analysis.
Countess 3 cell counter
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Countess 3 cell counter is a compact and easy-to-use instrument designed for accurate cell counting and viability analysis. It utilizes advanced imaging technology to provide quick and reliable cell enumeration results. The Countess 3 cell counter is a versatile tool suitable for a wide range of cell-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Chromium nuclei isolation kit, by 10x Genomics (1 mentions)
Novaseq 6000 sp, by Illumina (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Countess 3, by Thermo Fisher Scientific (1 mentions)
Ficoll solution, by GE Healthcare (1 mentions)
L glutamine, by Merck Group (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Lonza (1 mentions)
Hek293t, by Beyotime (1 mentions)
Cyquant mtt cell viability assay kit, by Thermo Fisher Scientific (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
11 protocols using countess 3 cell counter
1
Single Nuclei RNA Sequencing of Frozen Samples
2
Engrafting Human PBMCs in NSG Mice
3
Hematopoietic Cell Viability Analysis
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Following hematopoietic differentiation, floating suspension cells were collected and analyzed using a Countess 3 cell counter (ThermoFisher, Waltham, USA). Cell viability and cell number was measured after Trypan blue was added to testing samples.
4
Isolation and Characterization of PBMCs
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About 8–10 mL of peripheral blood was collected in an EDTA tube from 5 healthy volunteer donors between 24 and 45 years, 2 men, and 3 women, which did not present recent (a month before the blood collection) infection (such as respiratory syndromes, hepatic or gastrointestinal compromise), metabolic syndromes or cancer. Peripheral blood mononuclear cells (PBMCs) were isolated from blood using a 1.077 g/mL Ficoll solution (300× g/30 min/26 °C) (GE Healthcare®, Chicago, IL, USA). After two washes with 1X PBS, the cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM-Invitrogen®) with 10% fetal bovine serum (Gibco®) added, 1% L-Glutamine (Sigma®) and antibiotics. Cells obtained from each volunteer were cultured in 75 cm2 culture flasks (24 h/5% CO2/37 °C). The next day, the suspended lymphocytes were centrifuged in PBS 1X (200× g/10 min) and counted using the Countess 3 cell counter (Thermo Fisher®, Waltham, MA, USA). From the same culture flask, the adhered macrophages were removed with the aid of 2% trypsin, followed by centrifugation with 1X PBS (200× g/10 min) and counted (Countess 3/Thermo Fisher®). Lymphocytes and macrophages were plated (48-well plates/105 cells/well) in different cell culture schemes (see Section 2.4 ).
5
Bronchoalveolar Lavage Fluid Collection
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Rats were infected with ExoY+ (108) and monitored for over 48 h. Control and infected rats were then anesthetized with isoflurane gas (1%–3%) and once a surgical plane was established, as determined by a negative toe and tail pinch reflex, the trachea was cannulated, and a sternotomy was performed, removing the heart and lungs en bloc. The cannula was connected to tubing attached to a 60 mL syringe that was adjusted to provide 25 cmH2O filling pressure. PBS was allowed to flow into the lungs using the force of gravity until full (∼10 mL), at which point the tubing was disconnected and a syringe connected directly to the tracheal cannula to remove the PBS. This process was repeated three times to collect ∼30 mL of bronchoalveolar lavage fluid (BALF). BALF was quickly vortexed, and 1 mL was transferred into a separate tube for cell counting using a Countess 3 Cell Counter (Thermo Fisher) and bacterial cell counting. The remaining BALF was centrifuged at 500 g for 5–10 min. The supernatant was collected and sterilized via a 0.2 µm filter and frozen at −80° for later use. The remaining cell pellet was frozen at −80°C in full DMEM containing 20% FBS and 14% DMSO for analysis at a later date.
6
Viability Assessment of Transfection Reagents
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HEK293T cells were plated at a density of 7.5 × 104 cells per well in a 24-well plate and allowed to attach. Formulations (prepared as described above), 40 μg PEI 60K, or 75 μg exosomes were added directly to the culture media. Incubation was done in 1.5 mL culture media for 48 h, and then cells were harvested for the detection of viability. Cells were detached and washed with PBS before resuspension in culture media. Equal volumes of trypan blue and cell suspension were mixed briefly by pipetting and cell viability assessed using a Countess 3 Cell Counter (Thermo Fisher Scientific). Cell survival was calculated as a percentage of treated cells alive compared to untreated cells. Treatments were performed in biological triplicate. Three biological replicates were analyzed using a one-way ANOVA followed by Dunnett’s post hoc analysis, compared to the untreated control.
7
Trypan Blue Cell Viability Protocol
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Cell counts and viability were determined by trypan blue exclusion using a 1:1 mixture of cell suspension to 0.4% trypan blue solution (Invitrogen), and a Countess III cell counter (ThermoFisher).
8
Quantifying Cell Migration in Wound Healing
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Cell migration was measured in a wound healing assay. Briefly, BAECs plated in 6-well plates were transfected with siRNAs and allowed to reach 90% confluency over 48 h. Scratches on the monolayer were performed with a 200-μl pipette tip. Images of the wounded area were taken using a Zeiss Axio-Observer Z1 epifluorescent microscope (Zeiss Group, Oberkochen, Germany) (2.5×) at the beginning (t = 0) and after 16 h of migration. Wound closure was measured using the draw spline contour tool on Zen Blue.
Cell viability was determined by Trypan blue exclusion. Viable cells were manually counted using a hemacytometer or a Countess III cell counter (ThermoFisher Scientific, Waltham, MA, USA).
Cell viability was determined by Trypan blue exclusion. Viable cells were manually counted using a hemacytometer or a Countess III cell counter (ThermoFisher Scientific, Waltham, MA, USA).
9
Isolation and Enumeration of Murine Bone Marrow and Bone Cells
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Mice were weighed, and peripheral blood was obtained via submandibular vein puncture. Blood was collected in K2EDTA-coated microtainers, and automated blood count analysis was performed using a Scil Vet abc Plus+ counter (HORIBA Medical, Kyota, Japan). The mice were euthanized by cervical dislocation, and single-cell suspensions of bone marrow and bone fraction were prepared from the hind legs of mice as previously described.22 (link) In brief, hind legs were stripped from soft tissue, and bones were crushed and washed in PBS (Gibco) with 0.5% FCS. Cell suspensions were mechanically dispersed through a 40-µm nylon mesh cell strainer (Corning) and pelleted at 600g, followed by lysis of red blood cells using ACK lysis buffer (Lonza). Cells were subsequently washed and filtered using a 40-µm strainer. To determine bone marrow cellularity, single-cell suspensions, derived from 1 femur, were mixed 1:1 with Trypan Blue (Invitrogen), and the viable cell count was determined using a Countess 3 Cell Counter (Invitrogen). Remnants of crushed bones (bone fraction) were incubated with collagenase for 45 minutes at 37°C, washed with PBS, and filtered through a 40µm strainer. The remaining red blood cells in bone fraction were lysed using ACK lysis buffer (Lonza) and washed again with PBS, followed by another filtration through a 40-µm cell strainer.
10
GPCR Functional Assay in HEK293T Cells
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Pharmacological studies of GPCRs generally utilize mammalian cells to avoid the presence of endogenous GPCRs and achieve higher expression efficiency on the cell membrane. HEK293 and its derivative cell lines, such as HEK293T, as well as CHO cells, are the most commonly used cell lines in this field of research [19 (link),22 (link),26 (link),38 (link)]. In this study, we employed HEK293T cells as the research platform according to a method we established previously [39 (link)].
Human embryonic kidney cells HEK293T obtained from Beyotime Biotechnology (Shanghai, China) were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing a 10% fetal bovine serum in an incubator (37 °C and 5% CO2 humidified atmosphere). The cells were plated into 24-well plates and cultured for 24 h before assays. The Countess 3 cell counter (Invitrogen, Waltham, MA, USA) was used for cell counting.
Human embryonic kidney cells HEK293T obtained from Beyotime Biotechnology (Shanghai, China) were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing a 10% fetal bovine serum in an incubator (37 °C and 5% CO2 humidified atmosphere). The cells were plated into 24-well plates and cultured for 24 h before assays. The Countess 3 cell counter (Invitrogen, Waltham, MA, USA) was used for cell counting.
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