Timed matings were set overnight between sets of transgenic βH1-eGFP male and two wild-type ICR females. The presence of vaginal plugs was assessed the following morning. The day in which the plug was found was considered E0.5. The exact developmental stage of embryos was assessed by somite counts and morphological features47 (link). The dissected tissues were trypsinised, single cell suspensions were filtered through Filcon (Becton Dickinson) and further characterised by FACS analysis or tissue culture.
Filcon
Manufactured by BD
Sourced in United States
Filcon is a laboratory filtration equipment used for the separation and purification of liquid samples. It is designed to efficiently filter and remove particulate matter, suspended solids, or other contaminants from liquid solutions.
Automatically generated - may contain errors
Lab products found in correlation
Medimachine, by BD (2 mentions)
Medimachine system, by BD (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Merck Group (1 mentions)
Rompun, by Bayer (1 mentions)
Zoletil, by Virbac (1 mentions)
Its 3, by Merck Group (1 mentions)
Lab tek, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Collagenase a, by Roche (1 mentions)
70 m cell strainer, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Pe anti human cd8, by BioLegend (1 mentions)
Anti human cd4 pe cy7, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
16 protocols using filcon
1
Embryonic Staging via Transgenic Mice
2
Murine Immunological Tissue Harvesting
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For mice sacrifice, animals were anesthetized intraperitoneally with 100 µl of isotonic solution containing 1 mg of Zoletil (Virbac, Milano, Italy) and 200 µg Rompun (Bayer, Milano, Italy) to collect spleens, inguinal lymph nodes (ILN) and lower genital tract (LGT). Splenocytes and cells from ILN were purified from organs squeezed on filters as previously described [7] .
LGT was minced with sterile scissors and pieces of 1 mm were kept in RPMI supplemented with 1 mg/ml Collagenase (Sigma, Milano, Italy) for 90 minutes at 37°C. Subsequently the medium was filtered through a 70 µm cell strainer and with a 50 µm filcon (Becton Dickinson, Milano, Italy).
Blood samples for detection of HSV-specific T cells were collected from retro-orbital plexus in heparinized tubes and, after red blood cells lysis, stained with dextramers and conjugated antibodies.
LGT was minced with sterile scissors and pieces of 1 mm were kept in RPMI supplemented with 1 mg/ml Collagenase (Sigma, Milano, Italy) for 90 minutes at 37°C. Subsequently the medium was filtered through a 70 µm cell strainer and with a 50 µm filcon (Becton Dickinson, Milano, Italy).
Blood samples for detection of HSV-specific T cells were collected from retro-orbital plexus in heparinized tubes and, after red blood cells lysis, stained with dextramers and conjugated antibodies.
3
Primary Cell Culture Isolation
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Tissue biopsies were processed to obtain fresh cells as explained earlier [7] (link). Briefly, tissue was washed once with phosphate buffered saline (PBS; PAA, UK) before being minced and incubated in collagenase A (5 mg/ml, Roche Diagnostics, UK) at 37°C for 16 hrs. The cell suspension was then passed through a 70 µm cell strainer (BD Biosciences, USA) to remove any remaining tissue. Cells were then washed once with PBS and then passed through a nylon mesh filter (50 µm Filcon, BD Biosciences, USA) to be used immediately as fresh cells for FACS analysis and cell culture. Cells were maintained in culture up to 8 days in FibroLife medium (CellSystems Biotechnologie Vertrieb GmbH, Germany) supplemented with 2 mM L-glutamine, 100-units/ml penicillin, 100 mg/ml streptomycin and 1x ITS+3 (Sigma-Aldrich, UK). Cells were grown in 96-well plate (Corning, USA) and kept at 37°C/5% CO2. For immunofluorescence cells were also grown in 8-well chamber slides (LabTek, Sigma-Aldrich, UK).
4
Isolation and Surface Staining of Lymphoid Cells
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Immediately after mice were sacrificed, MLN and PP were placed in cold PBS. While on ice and moist at all times, single cell suspensions were made by squeezing the organs between the rough surfaces of two microscope slides. The cell suspensions were passed through 100 μm nylon filters (Filcon, BD Biosciences, Kgs. Lyngby, Denmark), spun down, re-suspended in PBS and transferred to 96-well plates. Cells were surface stained for CD4, CD3, CD11b, CD11c, CD103, and CD19, for 30 min using the appropriate antibodies (eBioscience, San Diego, CA, United States). Intracellular staining for FOXP3 was done after fixating the cells with Fixation/Permeabilization Buffer (stock concentrate diluted 1:4 with diluent; eBioscience) for 30 min, whereafter Permeabilization Buffer (eBioscience) diluted 1:10 with MilliQ water was added for 30 min. Analysis was performed using an Accuri C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI, United States).
5
Generation of Human T Cells from iPSCs
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A total of 30,000 cells per well of a 12-well plate were cultured on mitomycin C-treated OP9-hDLL1 in stromal culture medium with human cytokines as follow: 20 ng/mL FLT3L, 100 ng/mL SCF, 25 ng/mL IL-2, and 5 ng/mL IL-7 during the first 2 weeks and 10 ng/mL FLT3L, 25 ng/mL IL-2, and 5 ng/mL of IL-7 for the last 2 weeks. The cultures were transferred to fresh OP9-hDLL1 cells every 5 days after the first 10 days. For passaging, the cultures were treated with TrypLe (Thermo Fisher Scientific) and filtered through a 50-μm sterile Filcon (BD). Cells were analyzed after 1 month of culture using hCD45-PeCy5.5 (eBioscience), anti-human CD8-PE (BioLegend), and anti-human CD4-PeCy7 (eBioscience).
6
Single-cell sequencing of pancreatic progenitors
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Cells differentiated at day 13 were harvested with TrypLE Select as described above, resuspended in Stage 5 medium supplemented with 10 μM Y-27632, filtered using a 50 μM sterile Filcon (BD Biosciences) and sorted using a FACSAria Fusion cell sorter (BD) directly into 2 x 384-well plates with ERCC spike-ins (Agilent), reverse transcription primers and dNTPs (both Promega), according to the gating shown in Figure S10 B. Repartition was as follow: 216 cells for each NKX6-1-GFP+, NEUROG3-mCherry+ and NKX6-1-GFP+/NEUROG3-mCherry+ population and 104 cells for the negative population. Single cell sequencing was performed according to the Sort-seq method68 (link) by Single Cell Discoveries. Briefly, Sequencing libraries were generated with TruSeq small RNA primers (Illumina) and sequenced paired-end at 60 and 26 bp read length, respectively, on the Illumina NextSeq. Reads were mapped to the human GRCh38 genome assembly. Sort-seq read counts were filtered to exclude reads with identical library-, cell- and molecule barcodes. UMI counts were adjusted using Poisson counting statistics.68 (link)
7
Isolation of Skin and Lymph Node Cells
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Using an insulin microneedle, 8 mm skin biopsies were injected 5–10 times with Liberase TL (250 μg/mL)-DNase (500 μg/mL) solution, placed in 1 ml of Liberase TL-DNase solution in a 24-well plate (dermal side down), and then incubated for 2 h at 37°C in a cell culture incubator (5% CO2). To stop the enzymatic action at the end of the incubation period, 500 μL of DNase working solution and 75 μl of 0.1 M EDTA were added. Tissues were placed in a Medicon tissue grinder (BD Biosciences) and mixed for 8 min in a Medimachine (BD Biosciences). Cell suspensions were filtered using 50 μm syringe filter (Filcon, BD Biosciences), washed and counted.
Skin draining lymph nodes were harvested in Petri Dishes containing 1 mL RPMI 1640. Then, 1 mL of Liberase TM (0.52 U/mL)/DNase I (50 μg/mL) in RPMI 1640 were added to each Petri Dish. Each LN was flushed using an insulin microneedle and incubated for 20 min at 37°C. Two hundred fifty microliter of solution of EDTA at 100 mM were added to each Petri Dish to stop the reaction. Lymph node cell suspensions were then obtained by subsequent tissue dissociation and filtration through a cell strainer (100 μm), washed and counted.
Skin draining lymph nodes were harvested in Petri Dishes containing 1 mL RPMI 1640. Then, 1 mL of Liberase TM (0.52 U/mL)/DNase I (50 μg/mL) in RPMI 1640 were added to each Petri Dish. Each LN was flushed using an insulin microneedle and incubated for 20 min at 37°C. Two hundred fifty microliter of solution of EDTA at 100 mM were added to each Petri Dish to stop the reaction. Lymph node cell suspensions were then obtained by subsequent tissue dissociation and filtration through a cell strainer (100 μm), washed and counted.
8
Quantification of mCD44v6 and MTA1 Expression
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Tissue samples wetted with 1 ml 1X PBS were mechanically disaggregated by placing them into a Medicon (BD Biosciences) and inserted in the Medimachine (BD Biosciences) for 45 sec at 100 rpm. Cell suspensions were filtered using a Filcon (BD Biosciences), washed twice with 1X PBS and divided at a concentration of approximately 1×107 cells/ml into aliquots for staining. For detection of mCD44v6 positive cells, cell aliquots were incubated with CD44v6 (Bio-Rad MCA1730, dilution 1:500) for 1 h at room temperature (RT) in an orbital shaker at low speed. Cells were then washed and the percentage of cells expressing mCD44v6 was detected following a 60 min incubation with a goat anti-mouse IgG1: FITC secondary antibody (Bio-Rad STAR132F; dilution 1:100).
For intracellular labelling, cells fixation-permeabilization was performed using 1% paraformaldeyde-1% saponin in 1X PBS for 20 min at 4°C before cells were incubated with MTA1 antibody (SantaCruz Biotechnology SC-17773 dilution 1:500) and stained with a Polyclonal Goat anti-mouse-RPE Goat F(ab)2 secondary antibody (Dako R0480, dilution 1:100). Unstained cells were processed in parallel and included as references during analysis as well as cells stained only with the secondary antibodies. Flow cytometry analysis was performed using a Coulter Epics XL-MCL Flow Cytometer and its integrated SYSTEM II™ Software.
For intracellular labelling, cells fixation-permeabilization was performed using 1% paraformaldeyde-1% saponin in 1X PBS for 20 min at 4°C before cells were incubated with MTA1 antibody (SantaCruz Biotechnology SC-17773 dilution 1:500) and stained with a Polyclonal Goat anti-mouse-RPE Goat F(ab)2 secondary antibody (Dako R0480, dilution 1:100). Unstained cells were processed in parallel and included as references during analysis as well as cells stained only with the secondary antibodies. Flow cytometry analysis was performed using a Coulter Epics XL-MCL Flow Cytometer and its integrated SYSTEM II™ Software.
9
Isolation and Fractionation of Testicular Cells
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Isolation of testicular cells was performed according to previously published protocol [26 (link)] with minor modifications. Briefly, testes were cut into
2–3 mm3 pieces after removal of the tunica albuginea. These pieces were then placed into a disposable disaggregator Medicon with 1 ml of ice-cold PBS and processed for 50 sec in
Medimachine System (BD Biosciences). Cell suspension was recovered from Medicon unit using disposable syringe, and subsequently, filtered through 50 μm Filcon (BD Biosciences) and 25 μm
nylon mesh. Further, cells were washed once and resuspended in ice-cold PBS. Nuclear and cytoplasmic fractions of testicular cells were separated using NE-PER Nuclear and Cytoplasmic
Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two set of primers were used to check the quality of the two fractions as previously described
[27 (link), 28 (link)]. Gapdh (in5-ex6) was amplified to detect immature mRNA in the nucleus, and
Gapdh (ex5-ex6) was used to identify mature mRNA in the cytoplasm.
2–3 mm3 pieces after removal of the tunica albuginea. These pieces were then placed into a disposable disaggregator Medicon with 1 ml of ice-cold PBS and processed for 50 sec in
Medimachine System (BD Biosciences). Cell suspension was recovered from Medicon unit using disposable syringe, and subsequently, filtered through 50 μm Filcon (BD Biosciences) and 25 μm
nylon mesh. Further, cells were washed once and resuspended in ice-cold PBS. Nuclear and cytoplasmic fractions of testicular cells were separated using NE-PER Nuclear and Cytoplasmic
Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two set of primers were used to check the quality of the two fractions as previously described
[27 (link), 28 (link)]. Gapdh (in5-ex6) was amplified to detect immature mRNA in the nucleus, and
Gapdh (ex5-ex6) was used to identify mature mRNA in the cytoplasm.
10
Drosophila Germline Stem Cell Isolation and RNA Extraction
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The following Drosophila stocks were used in this study: w1118, bamΔ86 (McKearin & Spradling, 1990 (link)), bgcn20093 and bgcn20915 (Jin et al, 2008 (link)), UASp-tkvM1 (Casanueva & Ferguson, 2004 (link)), and nos-gal4 (Van Doren et al, 1998 (link)). Flies were maintained and crossed at room temperature on standard cornmeal/molasses/agar media unless otherwise specified. For total RNA isolation, fresh ovaries were dissected in the ice cold Grace Medium.
The vasa-GFP; hs-bam bam∆86/bam∆86 GSC line was established according to the published procedures (Niki et al, 2006 (link)). The GSCs cultured in 150-mm culture dishes were dissociated in StemPro Accutase Cell Dissociation Reagent (#A11105-01; Thermo Fisher Scientific) for 5 min and collected by centrifugation at 700g, 4°C for 5 min. The cell pellet was resuspended in PBS, passed through a 221/2 G needle five times, and filtered with 70-μm Filcon (340605; BD). GFP-positive GSCs and GFP-negative SCs were purified by sorting dissociated single cells at 20 ψ with 100 μm tip (InFlux; BD) immediately into TRIzol LS Reagent (10296028; Thermo Fisher Scientific) for total RNA isolation. Before RNA isolation, spike-in RNA standards from ExiSEQ NGS spike-in kit (800100; ExiSEQ) were added to the samples.
The vasa-GFP; hs-bam bam∆86/bam∆86 GSC line was established according to the published procedures (Niki et al, 2006 (link)). The GSCs cultured in 150-mm culture dishes were dissociated in StemPro Accutase Cell Dissociation Reagent (#A11105-01; Thermo Fisher Scientific) for 5 min and collected by centrifugation at 700g, 4°C for 5 min. The cell pellet was resuspended in PBS, passed through a 221/2 G needle five times, and filtered with 70-μm Filcon (340605; BD). GFP-positive GSCs and GFP-negative SCs were purified by sorting dissociated single cells at 20 ψ with 100 μm tip (InFlux; BD) immediately into TRIzol LS Reagent (10296028; Thermo Fisher Scientific) for total RNA isolation. Before RNA isolation, spike-in RNA standards from ExiSEQ NGS spike-in kit (800100; ExiSEQ) were added to the samples.
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