Computational protocol: Simplified isolation and enrichment of spermatogonial stem-like cells frompubertal domestic cats (Felis catus)

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[…] Suspension of dissociated testicular cells (n=6, 2 × 106 cell/ml) was first layered onto a discontinuous PercollTM gradient as previously described [] with some modifications. The PercollTM layers containing SSC-like cells were subsequently plated onto laminin-coated dishes for 15 min. The SSEA-4+ and GFRα-1+ cells were determined. In addition, the attached cells were collected for a study of SSC-related gene expression (POU5F1, RET and ZBTB16) and a differentiation marker (KIT).Animals and sample preparation: Tomcats (aged between 1–2 years old) were used in this study. Cat testes were consentingly collected following routine castration at the Veterinary Public Health Division of the Bangkok Metropolitan Administration, Bangkok, Thailand. The testes were transported to the laboratory in saline solution supplemented with antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin) at room temperature. They were dissected from extraneous testicular tissues prior to use. The testes were divided for fixation with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) (Exp. 1) and for dissociation with enzymatic digestion (Exp. 2 and 3).Histology, immunohistochemistry and immunofluorescence: For histology and immunohistochemistry, testicular tissue was fixed with 4% (w/v) paraformaldehyde (BDH Prolabo, VWR, Poole, U.K.) in PBS at 4ºC (overnight). The fixed testes were embedded in paraffin and cut at a thickness of 4 µm. Hematoxylin and eosin staining was used to study the structures of the testis.To detect the expression of laminin, a 3-step indirect immunoperoxidase immunohistochemistry (IHC) was performed with a Leica Microsystems BOND-MAX System (Leica Microsystems, Bannockburn, IL, U.S.A.). Briefly, the epitopes of the antigen were retrieved with Bond Epitope Retrieval Solution 2 (Leica Microsystems) for 20 min at 100ºC. The slides were incubated with an anti-mouse monoclonal laminin antibody diluted 1:100 (NovocastraTM, Leica Microsystems) at 25ºC for 45 min. Post Primary Polymer (Leica Microsystems) was applied for 9 min and followed with Polymer Poly-HRP IgG (Leica Microsystems) for 7 min. Mouse IgG (PP54, Millipore, Darmstadt, Germany) was used instead of the primary antibody for the negative control.For immunofluorescence (IF) on paraffin-embedded sections, the epitope was unmasked by microwave treatment at 900 watts for 15 min in citric acid buffer (BDH Prolabo; pH=6.0) supplemented with 0.03% (v/v) Triton X-100. Nonspecific staining was blocked using 3% (w/v) bovine serum albumin (BSA) in PBS. The sections were firstly incubated at 4ºC overnight with either mouse monoclonal SSEA-4 (1:200, Abcam, Cambridge MA, U.S.A.) or mouse monoclonal GFRα-1 (1:200, sc-10716, Santa Cruz Biotechnology, Dallas, TX, U.S.A.). They were further incubated with the corresponding secondary antibody (goat anti-mouse IgG TRITC, 1:200). Subsequently, the sections were co-stained with the primary antibody (rabbit polyclonal DDX-4, 1:100, Abcam) and followed by goat anti-rabbit IgG FITC (1:100, Abcam). For the negative control, the staining procedures were identically performed as described above except that the primary antibodies were replaced with mouse IgG (PP54, Millipore, Darmstadt, Germany) or rabbit IgG (PP64, Millipore). The co-expression of SSEA-4/DDX-4 and GFRα-1/DDX-4 markers was visualized with a fluorescence microscope (BX5, Olympus, Tokyo, Japan).The photomicrographs obtained from histology, IHC and IF were recorded using the cellSens software (Olympus) and Adobe Photoshop CS6 Version 13.0.1 (Adobe Systems, San Jose, CA, U.S.A.).For digested testicular cells, the cell suspension or attached cells were fixed with 4% (w/v) paraformaldehyde in PBS for 24 hr at 4ºC and then labeled with primary and secondary antibodies as mentioned above (SSEA-4/TRITC, GFRα-1/TRITC). The fixed cells were also blocked with 3% (w/v) BSA in PBS to reduce nonspecific background staining.Viability test and enrichment of SSC-like cells: Testes were enzymatically dissociated to obtain single testicular cells as previously described []. The testicular cells were examined for viability in terms of esterase enzyme activity (calcein AM staining) and plasma membrane integrity (ethidium homodimer-1, Molecular Probes, Life Technologies, Carlsbad, CA, U.S.A.).For differential plating selection, the culture dishes were first coated with either 20 µg/ml laminin or 0.1% (w/v) gelatin at 37ºC for 4 hr before cell plating. The dissociated testicular cells (0.5 × 106 cells/cm2) were plated onto laminin- or gelatin-coated dishes. The cells were further incubated for 15, 30 and 60 min, and then, the samples were fixed and collected for immunostaining. Culture was performed at 37ºC in a moisture incubator with 5% CO2 in air.PercollTM gradient density centrifugation was performed by layering the cell suspension (2 × 106 cells/ml) onto 30%, 45%, 60% and 90% (v/v) isotonic PercollTM solution, respectively. The cells were then centrifuged at 800 × g for 30 min at 25ºC. The thin layers of cell suspension at interfaces between the two concentrations of PercollTM were gently collected.RT-PCR for SSC-related gene expression: The attached testicular cells were collected and extracted to obtain total RNA using an Absolutely RNA Nanoprep Kit (StratageneTM, Agilent Technologies, Santa Clara, CA, U.S.A.). The total RNA (2 ng/µl) was reversely transcribed using the SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad CA, U.S.A.) for cDNA synthesis (RT+). Removal of SuperScriptTM III reverse transcriptase was performed for the negative control (RT-). PCR was performed with RT+ and RT- cDNA using GoTag® Green Master Mix (Promega, Fitchburg, WI, U.S.A.). In brief, the PCR conditions consisted of denaturation (2 min at 95ºC); 30 cycles of 30 sec at 95ºC, 30 sec at the annealing temperature for each primer and 30 sec at 72ºC; and final extension (2 min at 72ºC). The PCR products were electrophoresed in 1% (w/v) agarose gel (Bio-Rad, Hercules, CA, U.S.A.) supplemented with 5% (v/v) RedSafeTM Nucleic Acid Stain Solution (iNtRON Biotechnology, Gyeonggi-do, South Korea) in TBE buffer. The products were detected by a Gel Documentation system (Syngene, Cambridge, U.K.).The primers and annealing temperatures used in this study were as follows: POU5F1 (5′-TGAGAGGCAACCTGGAGAAC-3′ and 5′-AACCACACTCGGACCACATC-3′, 55ºC, 112 bp, accession number: EU366914), RET (5′-TGTGCATGACTACAGGCTGG-3′ and 5′-CCTGCTCACAGTGAAGGTGT-3′, 63ºC, 193 bp, accession number: XM_003994195.1), ZBTB16(5′-GCAAGAAGTTCAGCCTCAAGC-3′ and 5′- GCTTGATCATGGCCGAGTAGTC-3′, 63ºC, 119 bp) [] and KIT(5′-TCCTGCT CCGCGTCCAGACA-3′ and 5′-CTTGCCCTTCCGGTCCGCAG-3′, 60ºC, 533 bp) []. GAPDH was used as a housekeeping gene [].Statistical analysis: The percentages of SSEA-4+ and GFRα-1+ testicular cells and viability were expressed as the mean ± SEM. The data were analyzed with SPSS Statistics Version 20.0.0 (IBM Corporation, Armonk, NY, U.S.A.). The statistical differences between the groups were tested by analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Differences between values with P<0.05 were considered statistically significant. […]

Pipeline specifications

Software tools cellSens, SPSS
Applications Immune system analysis, Miscellaneous
Organisms Felis catus