Mouse scRNA-seq and cell hashing with unique barcoded antibodies (BioLegend) were processed using Chromium Single Cell Gene Expression 3′ v3 Library and a Gel Bead kit following the manufacturer’s instructions (10x Genomics, PN-1000075). Cells were counted and checked for viability using a Vi-CELL XR cell counter (Beckman Coulter), and then injected into microfluidic chips to form gel beads-in-emulsion in a 10x Chromium instrument. Reverse transcription was performed on the gel beads-in-emulsion, and products were purified and amplified. DNA from antibody-derived tags was separated from cDNA based on size selection using SPRIselect beads (Beckman Coulter, B23318). Expression libraries and antibody-derived tag libraries were generated and profiled using a Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified with a Kapa Library Quantification kit (Roche, 07960255001). All libraries were sequenced using HiSeq4000 and NovaSeq (Illumina)
Vi cell xr cell counter
Manufactured by Beckman Coulter
Sourced in United States, Germany, United Kingdom
The Vi-Cell XR cell counter is a fully automated cell viability analyzer that provides fast and accurate cell counts and viability measurements. It utilizes trypan blue dye exclusion to distinguish viable and non-viable cells, and can analyze a wide range of cell types. The Vi-Cell XR delivers reliable results with minimal user intervention.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (7 mentions)
Kapa library quantification kit, by Roche (7 mentions)
Hiseq 4000, by Illumina (6 mentions)
Chromium single cell 3 library and gel bead kit v2, by 10x Genomics (4 mentions)
Calcusyn software, by Biosoft (3 mentions)
Unique barcoded antibodies, by BioLegend (2 mentions)
Novaseq, by Illumina (2 mentions)
Spriselect beads, by Beckman Coulter (2 mentions)
Tryple select, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Transwell permeable support, by Corning (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
70 μm cell strainer, by BD (2 mentions)
Gel bead kit, by 10x Genomics (1 mentions)
Lipofectamine 2000, by Cell Biolabs (1 mentions)
Virabind aav purification mega kit, by Cell Biolabs (1 mentions)
Quicktiter aav quantitation kit, by Cell Biolabs (1 mentions)
L gln, by Thermo Fisher Scientific (1 mentions)
Anti clumping agent, by Thermo Fisher Scientific (1 mentions)
83 protocols using vi cell xr cell counter
1
Single-cell RNA-seq and Cell Hashing
2
Single-cell RNA-seq of Purified CDP Population
3
Production of High-Titer AAV in 293FT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293FT cells were counted using a Vi-Cell XR Cell Counter (Beckman Coulter) and 1 × 107 cells were plated using DMEM media containing FBS without penicillin and streptomycin on a 15-cm tissue-culture dish. The following day, the cells were transfected using Lipofectamine 2000 with the AAV vector along with AAV-DJ and AAV-pHelper packaging vectors (Cell Biolabs) at a ratio of 1:1:1. The next day, fresh DMEM media with 10% FBS was added; 48 h later, the cells and media were collected and subjected to four freeze-thaw cycles by alternating between an ethanol dry ice bath and 37°C. Cell debris was removed by centrifugation and the supernatant was collected, passed through a 0.45-µm filter, aliquoted, and frozen at −80°C until use.
To generate high-titer AAV for in vivo experiments, AAV was generated as described above, but 10 15-cm tissue-culture dishes of 293FT cells were transfected with AAV, and all transfected cells were pooled when the virus was harvested. AAV virus was purified using Virabind AAV Purification Megakit (Cell Biolabs #VPK141) according to the manufacturer's instructions. AAV virus was titered using QuickTiter AAV Quantitation Kit (Cell Biolabs ##VPK145) according to the manufacturer's instructions.
To generate high-titer AAV for in vivo experiments, AAV was generated as described above, but 10 15-cm tissue-culture dishes of 293FT cells were transfected with AAV, and all transfected cells were pooled when the virus was harvested. AAV virus was purified using Virabind AAV Purification Megakit (Cell Biolabs #VPK141) according to the manufacturer's instructions. AAV virus was titered using QuickTiter AAV Quantitation Kit (Cell Biolabs ##VPK145) according to the manufacturer's instructions.
4
Optimized CHO Cell Cultivation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO-S (cGMP Banked, Cat. No. A13696-01), was purchased from Thermo Fisher Scientific (USA). CHO-K1 and S were maintained in CD-CHO medium (Thermo Fisher Scientific) supplemented with 8 mM L-Gln (Thermo Fisher Scientific, USA) and 0.2% Anti-Clumping Agent (Thermo Fisher Scientific, USA). Cells were grown in either TubeSpin Bioreactor 50 (TPP) tubes (TPP Techno Plastic Products, Switzerland) with a working volume of 15 mL or 125 mL shaking flasks (Corning, USA) with a working volume of 25–30 mL. The cell cultures in the tubes and shaking flasks were incubated at 37 °C, 7% CO2, and humidified air at a shaking speed of 250 and 140 rpm, respectively. The cells were passaged twice a week with a seeding density of 2 × 105 viable cells per mL. The cell cultures were mixed with trypan blue and counted by ViCELL XR Cell Counter (Beckman Coulter, Germany) for VCD and % viability measurement.
5
Isolation of CD4+ T Cells from Patient Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human lymphocytes were isolated from peripheral blood from patients with AS, RA, PsA, and SLE and healthy controls by Pancoll® density gradient centrifugation (PAN™-Biotech GmbH, Aidenbach, Germany). CD4+ T cells were purified by negative selection using the CD4+ T cell isolation kit. Where indicated, CD45RA+RO− naïve CD4+ T cells were magnetically sorted using CD45RA MicroBeads and the autoMACS pro device (all Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the sorted cell populations was verified by flow cytometry and was at least 96%. Viable cells were counted using the Vi-CELL XR cell counter (Beckman Coulter, Krefeld, Germany) or the automated cell counter CellCountess (Life Technologies GmbH, Darmstadt, Germany).
6
Cell Viability Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at 8 × 10 E4 cells per well in 6-well plates and at indicated time points counted using the automated ViCell-XR cell counter (Beckman Coulter). Cell viability was determined by trypan blue exclusion.
7
Proliferation Assay for T and NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferation was monitored using cell counts and trypan blue dye exclusion with a Vi‐Cell XR cell counter (Beckman Coulter) or a WST‐1 colorimetric assay (11644807001; Roche) and a Victor X5 Light Plate Reader (2030‐0050; PerkinElmer) with a 450 nm filter.
For the WST‐1 assay, freshly purified NK and γδ T cells were seeded at 4,000 cells per well and the MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells were seeded at 10,000 cells per well in 96‐well flat‐bottom plates in duplicate in complete RPMI medium. A total of 8,000 (for NK and γδ T cells) or 20,000 (MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells) x‐irradiated (50‐Gy) aAPCs were added, and unless otherwise stated, antibodies, cytokines, peptides, or medium were added in 100 µL volumes to adjust the final volume of the culture to 200 µL. Cultures were incubated for 7 d at 37°C and 5% CO2. After 1 week, 20 µL of the WST‐1 reagent was added to each well, and the plates were incubated for an additional 4 h at 37°C and 5% CO2. The absorbance was measured at 450 nm using a Victor X5 Light Plate Reader. Data were normalized by calculating the percentage of proliferation using the formula: (test OD 450 nm/maximum OD 450 nm) × 100.
For the WST‐1 assay, freshly purified NK and γδ T cells were seeded at 4,000 cells per well and the MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells were seeded at 10,000 cells per well in 96‐well flat‐bottom plates in duplicate in complete RPMI medium. A total of 8,000 (for NK and γδ T cells) or 20,000 (MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells) x‐irradiated (50‐Gy) aAPCs were added, and unless otherwise stated, antibodies, cytokines, peptides, or medium were added in 100 µL volumes to adjust the final volume of the culture to 200 µL. Cultures were incubated for 7 d at 37°C and 5% CO2. After 1 week, 20 µL of the WST‐1 reagent was added to each well, and the plates were incubated for an additional 4 h at 37°C and 5% CO2. The absorbance was measured at 450 nm using a Victor X5 Light Plate Reader. Data were normalized by calculating the percentage of proliferation using the formula: (test OD 450 nm/maximum OD 450 nm) × 100.
8
Culturing Human Mammary Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human mammary epithelial cells (Lonza, #CC-2551) were cultured in MEGM Mammary Epithelial Cell Growth Medium (Lonza, #CC-3150). Human MCF-10A mammary epithelial cells (ATCC, #CRL-10317) were cultured in MEBM medium (Lonza, #CC-3151) supplemented with bovine pituitary extract, human epidermal growth factor, hydrocortisone and insulin (Lonza, #CC-4136 MEGM SingleQuot Kit) and 100ng/mL cholera toxin (Sigma-Aldrich, #C8052). Human breast carcinoma DU4475 (ATCC, #HTB-123) and MDA-MB-468 (ATCC, #HTB-132) cell lines were cultured in RPMI medium with 10% fetal calf serum (FCS) and penicillin-streptomycin (all from Invitrogen). Human ductal breast T-47D (ATCC, #HTB-133) epithelial tumour cells were cultured in IMDM medium (Lonza, #12-722F) supplemented with 10% FCS and penicillin-streptomycin (all from Invitrogen). For cultivation in stirred encapsulated bioreactor cultures, T-47D was grown in a phenol red free RPMI medium (for details see Encapsulated Stirred Bioreactor Culture). Cell numbers and viability were analysed using a Vi-CellXR cell counter (Beckman Coulter) following the manufacturer’s instructions.
9
Vero Cell Expansion and Maintenance
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Vero cell line ATCC CCL-81.2 was obtained from the ATCC. Vero cells were maintained in static culture using OptiPRO™ Serum Free medium (Thermo Fisher Scientific) supplemented with 4 mM l -glutamine (Thermo Fisher Scientific) at 37 °C and 5% CO2 in a humidified incubator (Thermo Scientific). Cells were passaged seven times to create a working cell bank, which was used for all experiments described herein. For static cell growth, cells were passaged for 4 passages in Corning CellSTACK® using TrypLE™ Select (ThermoFisher Scientific) for cell detachment and Soybean Trypsin Inhibitor (STI; 1 g/L) for protease inactivation (Millipore Sigma). Cell concentration and viability were determined using the Vi-CELL™ XR cell counter (Beckman Coulter, USA). All static cell growth manipulations were performed in a closed system via sterile welds and pumps, except for the initial vials thaw operation.
10
RCP-Mediated Culture of HMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HMSCs (0.3 × 106 cells/well) were seeded into a six-well Falcon plate in 2 mL Mesen PRO medium. Three RCP concentrations (0.028, 2.8, and 28 μg/mL) were prepared and added to the culture medium; these concentrations were based on the amount of RCP (2.8 μg/mL) used to coat wells. Control cultures received the same volume of DPBS in place of the RCP solution. Cells were cultured and passaged every 7 days. At the end of the culture period, cells were collected using trypsin-ethylenediaminetetraaceticacid (Lonza, Basel, Switzerland) and were counted using a Vi-Cell XR cell counter (Beckman Coulter, Inc., CA). Three repeat cultures were assessed for each treatment condition, and cell proliferation curves were obtained. Population doubling (PD) at the nth passage (Pn) can be described as follows:
PD at Pn = PD at Pn− 1 + log2 (counted cells/seeded cells).
PD at Pn = PD at Pn− 1 + log2 (counted cells/seeded cells).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!