Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 1. Frequency of stem cells in DSC-fibroblast co-cultures. Dermal cells were cultured in stem cell medium for up to two weeks to form spheres. The spheres were enzymatically dissociated with Accutase™, and the percentage of DSCs (NGFRp75-positive cells) in the culture was determined via flow cytometry. (A) Boxplot displaying the median with 25th/75th percentiles. The cross (×) inside the box indicates the mean DSC frequency. Dots represent outliers. (B) The histogram shows the abundance distribution of DSC frequencies of individual donor cell strains divided into ranges of 5%. Results from 66 donors.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Cell Culture, Cytometry

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 3. Selective detachment of DSC-fibroblast co-cultures with 10–20% stem cells. Dermal cells were incubated with Accutase™for 1, 2, or 3 min or with trypsin-EDTA for 0.5, 1, or 2 min, and detached cells were collected. (A) Frequency of DSCs in samples measured using NGFRp75 staining. (B) Viability of total cells examined with propidium iodide. (C) Plot of recovery rate (x-axis) versus purity (y-axis) of detached cells at the individual incubation times. Purity: frequency of DSCs. Recovery rate: ratio of the absolute number of DSCs in the detached sample to the absolute number of DSCs before detachment. Dotted lines indicate the DSC frequency of control cells detached with Accutase™(light gray) or trypsin-EDTA (dark gray). Results from one representative donor cell strain.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Incubation, Staining, Control

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 5. Positive selection (labeling of DSCs). (A–C) EasySep™column-free positive selection with the EasySep™Human CD271 Positive Selection Kit II (StemCell Technologies). Sample size: n = 6. (D–F) MACS® automatic column-based positive selection with Neural Crest Stem Cell (NCSC) MicroBeads and the autoMACS® Pro Separator (Miltenyi Biotec). Program: Posseld2. Sample size: n = 13. Values are presented as mean ± SDs. Pre: initial sample, neg frac: fibroblast fraction, pos frac: DSC fraction, d11–12: 11–12 days of cultivation. Frequency of DSCs in the separate fractions was determined via flow cytometry analysis of NGFRp75 (y-axis of dot plots) and CD90 (x-axis of dot plots). Following separation, the enriched DSC fraction was cultivated in stem cell medium. After 11–12 days, the proportion of DSCs in culture was measured again via flow cytometry, and cells were additionally stained immunohistochemically for NGFRp75 (red) and CD90 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 200 µm.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Selection, Labeling, Cytometry, Staining

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 6. Overview of purity and recovery of DSCs after the individual selection methods. (A) Purity. Frequency of DSCs in the purified fraction after separation determined via flow cytometry analysis of NGFRp75. Values are presented as mean ± SDs. (B) Recovery rate. Ratio of the absolute number of DSCs in the purified fraction after separation to the absolute number of DSCs in the pre sample. Values are presented as mean ± SDs. (C) Plot of recovery rate (x-axis) versus purity (y-axis) of the individual selection methods. Sample size: a. n = 5; b. n = 3; c. n = 2; d. n = 3; e. n = 3; f. n = 2; g. n = 6; h. n = 13.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Selection, Cytometry

Journal: Nature protocols

Article Title: Dual detection of chromatin accessibility and DNA methylation using ATAC-Me

doi: 10.1038/s41596-021-00608-z

Figure Lengend Snippet: (a) Fragment distribution of libraries converted using the EZ Methylation-Gold Kit (gold solid line) and EZ Methylation-Lightning Kit (blue dotted line). Fragment distributions are representative of electropherograms visualized on Agilent Tape Station results files. y-axis displays normalized fluorescence units (FU). (b) Sequence complexity was calculated using Pre-seq57 for three example ATAC-Me libraries of H9 ESCs representing a range of sequenced DNA fragment complexities. (c) Genome Browser tracks surrounding the Nanog locus for three ATAC-Me examples of H9 ESCs are shown alongside tracks of whole genome methylation data from Xie et al. 201316 (gold track). Single CpG methylation levels are shown as vertical blue bars with height corresponding to fraction methylation (calculated as mCpG reads/total CpG reads). The read coverage of each CpG is represented as the height of the black bars. Reads normalized to library depth are shown in green to represent accessibility signal calculated for each sample. Hypomethylated regions (HMRs) were determined using the MethPipe software package and correspond to peaks called by Genrich software (available at https://github.com/jsh58/Genrich)48.

Article Snippet: BIOLOGICAL MATERIALS H9 human embryonic stem cells (ESCs) (gift from Gama Lab, Vanderbilt University; WiCell, cat. no. WA09; https://scicrunch.org/resolver/RRID:CVCL_9773).

Techniques: Methylation, Fluorescence, Sequencing, CpG Methylation Assay, Software

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: (A) NP swab diluents from 2 confirmed COVID-19 patients were pooled, and using the 2019-nCoV_N3 primer/probe set, the mixture was either (i) subjected to RNA extraction using the Qiagen QIAamp Viral RNA Mini Kit followed by subsequent testing by RT-qPCR (using the equivalent of 11.3 μl of swab diluent) or (ii) directly added to the RT-qPCR reaction, with or without a preheating step (5 minutes at 70°C, “NP sample + heat”). As a control, the indicated quantities of the CDC 2019-nCoV Positive Control SARS-CoV-2 synthetic RNA were spiked into M6 transport medium, purified using the QIAamp Viral RNA Mini Kit, and screened by RT-qPCR. NP swab samples from 7 additional donors were screened by direct RT-qPCR for SARS-CoV-2 RNA using the 2019-nCoV_N1 primer/probe set (B) or the 2019-nCoV_N2 primer/probe set (C), or for human RNase P RNA using the RNase P primer/probe set (D). NP swab samples from donors 1–4 were previously shown to contain SARS-CoV-2 RNA by standard clinical RT-qPCR, while donors 5–7 were negative. For each primer/probe set, 7 μl (A) or 3 μl (B–D) of NP swab diluent was tested in the RT-qPCR reaction per donor. For the N1 and N2 primer/probe sets, the fully synthetic SARS-CoV-2 RNA Control 2 from Twist Bioscience was loaded at serial 10-fold dilutions (A, 3 × 10 6 copies; B, 3 × 10 5 copies; C, 3 × 10 4 copies; D, 3 × 10 3 copies; E, 3 × 10 2 copies; F, 3 × 10 1 copies) as indicated in (B) and (C). NTC wells were included for each primer/probe set, and each was negative. For (B) and (C), the correlation coefficients ( R 2 ) of the standard curves were 0.999 and 0.995, respectively. The dashed line at cycle 40 in each graph indicates the limit of detection. CDC, Centers for Disease Control and Prevention; CT, cycle threshold; NP, nasopharyngeal; NTC, no template control; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: RNA Extraction, Quantitative RT-PCR, Positive Control, Purification, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection of SARS-CoV-2 RNA from NP swab diluent by direct RT-qPCR and the impact of heat and loading volume on assay sensitivity.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: Detection sensitivity of direct RT-qPCR versus standard RT-qPCR on NP swabs containing a range of SARS-CoV-2 viral RNA loads.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques:

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 150 NP swab samples representing high (CT values less than 20), intermediate (CT values of 20–30), or low (CT values of more than 30) SARS-CoV-2 RNA loads as determined by standard clinical RT-qPCR at the University of Washington in Seattle (aqua circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was extracted from 30 μl of NP swab diluent that had been previously heated at 95°C for 10 minutes, and RNA representing 3 μl of the original diluent was used in RT-qPCR (purple circles) to allow a head-to-head comparison with direct RT-qPCR on the same quantity of NP swab diluent. The limit of detection (CT of 40) is denoted with a dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the mean of CT values detected in the clinical RT-qPCR assay with primer sets N1 and N2. Samples are ordered by the latter mean. The full dataset for this experiment and controls are provided in . CT, cycle threshold; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: A total of 60 NP swab samples representing low loads (CT of 27–36) of SARS-CoV-2 RNA as determined by standard clinical RT-qPCR at UW in Seattle (purple circles) were analyzed by the indicated method. All assays used the 2019-nCoV_N2 primer/probe set. Direct RT-qPCR was performed on 3 μl of NP swab diluent after heating for 10 minutes at 95°C (green circles). In parallel, RNA was newly extracted from 200 μl of NP swab diluent (aqua circles) and processed with the UW LDT to control for the effect of freeze/thaw cycles. The limit of detection (CT of 40) is denoted with a red dashed line. Samples with CT values above this cutoff were considered negative for SARS-CoV-2 RNA. The fitted curves are LOESS (locally estimated scatterplot smoothing)–smoothed CT values, with 95% confidence intervals in gray, against the CT values detected in the 200-μl freshly extracted RT-qPCR assay with primer set N2. Samples are ordered by the CT value of freshly extracted 200-μl LDT samples. The full dataset for this experiment and controls are provided in . CT, cycle threshold; LDT, laboratory developed test; NP, nasopharyngeal; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; UW, University of Washington.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

doi: 10.1371/journal.pbio.3000896

Figure Lengend Snippet: RT-qPCR conditions.

Article Snippet: In , 7 μl of pooled NP swab diluent (heated to 70°C for 5 minutes, or not), or 5 μl of extracted RNA, was used as input material for the New England Biolabs Luna Universal Probe One-Step RT qPCR Kit (Cat #E3006S, lot #10066679) according to the Integrated DNA Technologies (IDT) recommendation for primers/probes (1.5 μl of primer/probe per reaction) using primer set N3 from IDT’s 2019-nCoV CDC Emergency Use Authorization Kits (20-μl reaction).

Techniques: