Fetal gonads were dissected into ice cold 0.4% BSA in PBS, washed once, and maintained on ice until ready for digestion. 0.4% BSA solution was removed and replaced with 100 μL of 0.25% Trypsin-EDTA (Fisher Sci, 25200056) per ovary pair, or 150 μL per testis pair. Samples were incubated in a 37 °C water bath for 30 min, with gentle pipetting every 10–15 min to facilitate the dissociation. After 30 min, DNase I (1 mg / mL) was added at a 1:10 dilution, and samples were incubated another 10 min at 37 °C. Samples were pipetted to ensure complete digestion, and then an equal volume of ice-cold FBS (Gibco, 10437028) was added to inactivate trypsin. To prepare for FACS sorting, Sytox Blue viability dye (Invitrogen, S34857) was added to samples at 1:1000 dilution, and then samples were filtered through a 35 μm filter into FACS tubes (Falcon, 352235).
Sytox blue viability dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sytox Blue is a cell-impermeant nucleic acid stain that selectively binds to the DNA of cells with compromised membrane integrity, such as dead or dying cells. It can be used to distinguish viable from nonviable cells in a population.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Facs tube, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Facs aria fusion cytometer, by BD (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Chromium single cell 3 v3, by 10x Genomics (1 mentions)
Accuprime taq hifi, by Thermo Fisher Scientific (1 mentions)
Lsrii instrument, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Anti cd16 32 antibody, by BioLegend (1 mentions)
Fortessa, by BD (1 mentions)
Fluorophore conjugated primary antibodies, by BioLegend (1 mentions)
8 protocols using sytox blue viability dye
1
Dissociation and Preparation of Fetal Gonad Cells
2
ROBO1-Positive Cell Isolation and Cloning
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SW480 cells were washed with 1X PBS-EDTA and incubated with TrypLE Express Enzyme (Gibco) for 5 min at 37°C before quenching with DMEM-C. Cells were centrifuged at 300xg for 5 min at 4°C, washed with FACS buffer (1% BSA in PBS) and centrifuged again before resuspending the cell pellet in enough FACS buffer for 1million cells/100μL. For subtype-specific isolation, antibody incubation was performed on ice in the dark for 30 min (ROBO1-PE; NBP2-75975PE was custom made using MAB71181 (clone 770502)). After staining, samples were diluted with FACS buffer and centrifuged at 500xg for 5 min at 4°C. Final cell pellets were resuspended in FACS buffer for 2 million cells/100μL, passed through a 35 μm filter cap, and SYTOX blue viability dye was added (1:10,000, Invitrogen) before sorting on a BD FACSAria Fusion cytometer. Sorted samples were collected into 15 mL conical tubes containing DMEM-C or 1% BSA/PBS for subculture or sorted directly into 96-well plates containing DMEM-C for single cell clonal derivations. Single cell clonal derivations were incubated at 37°C for one month before first passage. Passages 4-10 of clonally derived cells were used in the described experiments and mycoplasma tested every 3-5 months.
3
Isolation of Gonadal Cell Populations
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Fetal gonads were dissected into ice cold 0.4% BSA in PBS, washed once, and maintained on ice until ready for digestion. 0.4% BSA solution was removed and replaced with 100 μL of 0.25% Trypsin-EDTA (Fisher Sci, 25200056) per ovary pair, or 150 μL per testis pair. Samples were incubated in a 37°C water bath for 30 minutes, with gentle pipetting every 10-15 minutes to facilitate the dissociation. After 30 minutes, DNase I (1 mg / mL) was added at a 1:10 dilution, and samples were incubated another 10 minutes at 37°C. Samples were pipetted to ensure complete digestion, and then an equal volume of ice-cold FBS (Gibco, 10437028) was added to inactivate trypsin. To prepare for FACS sorting, Sytox Blue viability dye (Invitrogen, S34857) was added to samples at 1:1000 dilution, and then samples were filtered through a 35 μm filter into FACS tubes (Falcon, 352235).
4
Circadian Skin Cell Isolation Protocol
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Immediately after sacrificing a mouse with CO2, hair on dorsal skin was removed with an electric razor and Nair Hair Removal cream. After the dorsal skin was isolated from the body, fat and remaining blood vessels were scrapped away with a scalpel blade. A circular piece of skin was obtained with a 12mm biopsy punch, and minced into tiny pieces. The minced skin was then digested with 2mL of a solution consisting of 0.27% Collagenase IV (Sigma, C5138), 10mM HEPES (Fisher Scientific, BP310-100), 1mM Sodium Pyruvate (Fisher Scientific, BP356-100), and 5U/mL DNase I (Thermo Scientific, EN0521) at 37 °C for 1.5 hours. The suspension was then filtered with 70 μm and 40 μm cell strainers to obtain single cells. SYTOX blue viability dye (1:1000; Invitrogen, S34857) was added to the cell suspension and live cells were sorted out using FACS at the UCI Stem Cell Research Center.
Samples were collected every four hours for three days to generate in total of 18 samples, providing three biological replicates per circadian time point. The Chromium Single Cell 3’ v3 (10x Genomics) libraries were prepared and sequenced by the University of California Irvine Genomic High Throughput Facility with Illumina NovaSeq6000.
Samples were collected every four hours for three days to generate in total of 18 samples, providing three biological replicates per circadian time point. The Chromium Single Cell 3’ v3 (10x Genomics) libraries were prepared and sequenced by the University of California Irvine Genomic High Throughput Facility with Illumina NovaSeq6000.
5
Monitoring GFP Expression and Targeted Integration in K-562 Cells
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K-562 cells were assessed for GFP expression every 7 days for 28 days following electroporation. Flow cytometry was conducted on an LSRII instrument (Becton Dickinson) and data was analyzed using FlowJo software v10 (Becton Dickinson). Dead cells were excluded from analysis by abnormal scatter profile and exclusion based on Sytox Blue viability dye (Thermo Fisher Scientific).
Junction PCR to detect targeted integration was conducted using external genomic primers outside of the 48 bp homology region and internal primers complementary to the expression cassette. PCR was conducted using Accuprime HIFI Taq (Thermo Fisher Scientific). PCR products from bulk population were sequenced directly.
Junction PCR to detect targeted integration was conducted using external genomic primers outside of the 48 bp homology region and internal primers complementary to the expression cassette. PCR was conducted using Accuprime HIFI Taq (Thermo Fisher Scientific). PCR products from bulk population were sequenced directly.
6
Single-Cell FACS Isolation and Analysis
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Cultured cells were dissociated (TrypLE Express, Gibco, Thermo Fisher Scientific) and filtered. Single cell suspensions were then blocked (10% horse serum in PBS/0.5 mM EDTA, Gibco, Thermo Fisher Scientific) for 30 min prior to incubation with the directly conjugated primary antibodies (listed above) in FACS buffer (3% horse serum in PBS/0.5 mM EDTA) for 1 h at 4 °C. Following washing with FACS buffer, cell pellets were gently resuspended in FACS buffer (200 μL) containing SYTOX Blue viability dye (Thermo Fisher Scientific). Cell analysis was conducted using a FACS Canto II instrument (BD Biosciences) with compensation as required. Cell sorting was conducted using a FACS ARIAIII instrument (BD Biosciences) with compensation as required. Data were analyzed using FlowJo software (v10, BD Biosciences).
7
Immune Cell Profiling of Spleen and Skin
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Single‐cell suspension of freshly isolated splenocytes and total skin was prepared as described earlier (Novoszel et al, 2021 (link)) and subsequently blocked with anti‐CD16/32 antibody (BioLegend). Samples were stained with following fluorophore‐conjugated primary antibodies (Biolegend) for 30 min at 4°C: Ly6G, CD11b, CD11c, CD19, F4/80, MHC class II, Ly6C, and CD3ε (spleen) and B220, BST‐2, CD3ε, CD11c, CD11b, CD19, F4/80, Ly6C, Ly6G, MHC class II, TCR γδ, and XCR1 (skin, two panels; see Appendix Table S2 ). After incubation, cells were washed, filtered, and stained with SYTOXblue viability dye (Thermo Fisher) according to the manufacturer's recommendation to exclude dead cells. Flow cytometry was performed on a Fortessa (BD Bioscience) and analyzed by FlowJo v10 software.
8
Determination of Cell Viability
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The percentage of RFP1 negative or DsRed2 positive cells was determined by flow cytometry. Cells were washed with phosphate buffered saline (PBS) and then incubated with 1 µM Sytox Blue viability dye (Thermo Fisher Scientific) in PBS containing 0.05% bovine serum albumin (BSA) at room temperature for 30 minutes before measurement on a SRII flow cytometry (Becton Dickinson, Mountain View, CA, USA), with analysis by FlowJo software (Tree Star, San Carlos, CA, USA).
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