Dissociated periodontal cells harvested from P25 Col1(2.3kb)-GFP;PTHrP-creER;R26R-tdTomato molars were pooled. Cell sorting was performed using a six-laser Sony Synergy SY3200 (Ex.350/405/488/561/594/685nm) high-speed cell sorter with a 100-μm nozzle. Col1a1(2.3kb)-GFP+ cells including tdTomato+ cells were directly sorted into ice-cold DPBS/1% FBS, pelleted by centrifugation and resuspended in 30 μl DPBS/1% FBS using wide-bore pipettes. Cell numbers were quantified by Countless II automated Cell Counter (ThermoFisher) before loading onto the Chromium Single Cell 3’ microfluidics chip (10× Genomics Inc., Pleasanton, CA). cDNA libraries were sequenced by Illumina HiSeq 4,000 using two lanes and 50 cycle paired-end read, generating a total of ~ 770 million reads. The sequencing data was first pre-processed using the 10× Genomics Cell Ranger software. For alignment purposes, we generated and used a custom genome fasta and index file by including the sequences of mCherry to the mouse genome (mm10). The scRNA-seq dataset presented herein have been deposited in the National Center for Biotechnology Information (NCBI)’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE120108 and GSE168450. The dataset GSE120108 has been published previously (2 (link)).
Countless 2 automated cell counter
Manufactured by Thermo Fisher Scientific
The Countess II Automated Cell Counter is a compact and efficient device designed to rapidly and accurately count cells in a variety of samples. It utilizes automated cell detection and analysis to provide reliable cell counts, viability data, and other cellular metrics. The Countess II is a versatile tool suitable for use in various laboratory settings.
Automatically generated - may contain errors
4 protocols using countless 2 automated cell counter
1
Isolation and Sequencing of Periodontal Cells
2
Oral Liquid-Based Cytology Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OLBC samples were obtained from all participants by oral medicine specialists using a defined protocol as outlined in our previous reports.13 , 15 A liquid‐based cytology (LBC) slide was prepared for each OLBC sample using the ThinPrep® 2000 processor (Hologic Inc., MA) according to the manufacturer's protocol.
The Countless II automated cell counter (ThermoFisher, MA) was then used to measure cell yields in each OLBC sample after preparing LBC slides. Any sample with at least 1 × 106 cells was considered adequate for further cell‐block preparation. The remaining cells in each ThinPrep® PreservCyt vial were processed to prepare an individual paraffin cell‐block for each case using the thromboplastin‐plasma method.15 The quality of each cell‐block was evaluated in terms of cellular morphologic preservation by visually assessing 4 μm hematoxylin and eosin‐stained sections.
The Countless II automated cell counter (ThermoFisher, MA) was then used to measure cell yields in each OLBC sample after preparing LBC slides. Any sample with at least 1 × 106 cells was considered adequate for further cell‐block preparation. The remaining cells in each ThinPrep® PreservCyt vial were processed to prepare an individual paraffin cell‐block for each case using the thromboplastin‐plasma method.
3
Isolation and Sequencing of Periodontal Cells
4
Single-Cell Sequencing of tdTomato+ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both male and female mice from the same litter were used to pool FACS-isolated tdTomato+ cells. Cell sorting was performed using a four-laser BD FACS Aria III (Ex.407/488/561/640 nm) high-speed cell sorter with a 100 µm nozzle. tdTomato+ cells were directly sorted into ice-cold DPBS/10% FBS, pelleted by centrifugation, and resuspended in 10 µl DPBS/1% FBS. Cell numbers were quantified by Countless II Automated Cell Counter (Thermo Fisher) before loading onto the Chromium Single Cell 3′ v3.1 or Single Cell ATAC v2 microfluidics chips (10x Genomics Inc., Pleasanton, CA) following the manufacturer’s recommended protocols. cDNA or gDNA libraries were sequenced by Illumina NovaSeq 6000. The sequencing data were first preprocessed using the 10X Genomics software Cell Ranger. For alignment purposes, we generated and used a custom genome fasta and index file by including the sequences of tdTomato-WPRE to the mouse genome (mm10).
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!