Micropipette

Males were weighed and injected in the dorsal musculature with 2 international units per gram (IU/g) of body weight with human chorionic gonadotropin (hCG) (BioVendor Laboratory Medicine Inc., Lot # RU-82209-4, Ashville, NC, USA) diluted in phosphate buffered saline (PBS) (Gibco, Ref # 10010-031, Grand Island, NY, USA) at a concentration of 2 IU/µL. Animals were returned to their tanks and collected again after 18–43 h. Animals were anesthetized in 0.15% tricaine methanesulfonate (MS-222 Syncaine, Syndel, Ferndale, WA, USA) that was diluted with aquarium water and buffered to a pH of 6.5–7.5 with sodium hydroxide (1.0 N standardized solution, VWR Chemicals, Radnor, PA, USA) (Supplementary File S1). When animals lost righting reflex after being flipped upside-down (about 15–20 min), they were removed and placed on a firm, inclined surface.
The abdomen and cloaca were rinsed with deionized (DI) water followed by Hanks’ Balanced Salt Solution at 200 mOsm/kg (HBSS200; pH 7.4) (Supplementary File S1). The sides and abdomen of the animals were massaged with gentle pressure from anterior to posterior toward the cloaca [9 (link)] (Figure 1A, Video S1). Massage motions were repeated, and sperm samples were collected using a micropipette (p1000 µL; Gilson Pipetman) (Figure 1A) and transferred to 1.5 mL centrifuge tubes. When necessary, larger animals were held with two hands when performing massages to better control pressure above the cloaca (Video S1). Urine was discharged with highly diluted sperm prior to the release of relatively concentrated sperm, and thus, the spermic urine (relatively clear liquid) and sperm fluid (opaque and white liquid) were collected in separate tubes. The time it took to anesthetize animals, collect sperm, and evaluate quality was recorded to identify potential bottlenecks during collection with limited personnel. The concentration (sperm/mL), volume (µL), and motility (described below) of collected sperm fluid were recorded, and the total number of sperm collected was calculated to evaluate relationships between animal size and sperm production.

Sperm were activated by gently mixing with a Gilson micropipette 1 μL of ejaculate preserved in ZSI with 10 μL of aged tap water. Three microliters of samples were then quickly placed in separate wells on a 12-well multi-test slide and covered with an 18 × 18 mm coverslip. Both slide and coverslip were previously coated with 1% polyvinyl alcohol (Sigma–Aldrich), to avoid sperm sticking to the glass slide Sperm velocity was measured using a CEROS Sperm Tracker (Hamilton Thorne Research, Beverly, MA, USA) in the first 10 s after activation. The measurement was repeated on three subsamples per male, leading to a repeatability of 0.68 ± 0.1422, and mean values were used for statistical analyses. Mean velocity measurements were based on 280 ± 169 (mean ± s.d.) sperm tracks per male. Since sperm velocity parameters (VAP: average path velocity, VSL: straight-line velocity, and VCL: curvilinear velocity) were highly correlated (all Pearson R > 0.88; all p < 0.001), only VAP was considered in the following analyses. The measurement was repeated twice per male, yielding a repeatability of 0.68 ± 0.14, and mean values were used for statistical analyses.

Balb/c mice were divided into three groups (n = 5/group): control, E10 and E11. The control group received 200 μL of apyrogenic water, and the groups were designated as E10, and E11 according to the extract received in a dose of 5 mg/kg, p.o. The animals were anesthetized intramuscularly with 0.4 mL of 2% xylazine chloridrate (20 mg/kg) and 5% ketamine chloridate (25 mg/kg). With a micropipette (Gilson), 10 μL of LPS solution (1 mg/mL sterile PBS) was given by the aerogenic route using nasal instillation. This induction was done on three consecutive days (Figure 2). One day before the first LPS application, the control, E10, and E11 treatment was started. This treatment continued for four days, and the sacrifice of the animals was performed 24 hours after the last application of LPS, when the bronchoalveolar lavage was performed as mentioned above [26 ].

Live or fixed tumor cells enriched from blood by ISET^®, collected without sticking them to the filter as described in section 5, and transferred in suspension to a cell culture plate could be gently picked up manually and individually with a micropipette (Gilson P2) under an inverted Olympus CK30-F2000 microscope at the x10 objective and transferred to an individual 0.2 mL PCR tube.
Single cells’ proteins were lysed so as to release the DNA using our in house protocol (in 15 μL, 100 mM TrisHCl pH 8 and 400 μg/mL of proteinase K incubated for 2 hours at 60°C followed by proteinase K inactivation for 5 min at 94°C). The single-cell DNA was then pre-amplified by combining our lysis protocol and by adapting it to single-cell whole genome amplification (WGA) commercial kits: MALBAC (Yikon Genomics, China); Picoplex (Rubicon Genomics and New England BioLabs, USA); GenomePlex (Sigma-Aldrich, USA). Amplified DNA was then purified using the DNA Clean & Concentrator™-5 kit (Zymo Research, Germany) and quantified by Quant-iT™ PicoGreen^® dsDNA Assay Kit (Thermo Fisher Scientific, USA). The WGA products' quality was assessed by PCR as described elsewhere [40 (link)].
For NGS single-cell analysis using the Illumina approach, 250 ng of the amplified DNA was used to prepare libraries for high-throughput sequencing with the Nextera Exome Enrichment Kit (Illumina, USA) following the supplier’s protocol. A fraction of the library DNA from the pre-capture step was used for whole genome analysis. The libraries were sequenced on a HiSeq2000 (Illumina) in 2 x 100 bp paired-end runs. Data were mapped against the human genome and the number of reads per chromosome was calculated.
In order to perform single-cell targeted theranostic mutations' NGS analysis, we used the ThermoFisher approach, 10 ng of purified WGA DNA was used to generate 207 amplicons libraries including 50 oncogenes and tumor suppressor genes through the use of the optimized primer pool “Hotspot cancer panel v2” and Ion Torrent™ Library Kit V2 (ThermoFisher, USA), according to manufacturer’s recommendations. Briefly, Ion adapters and barcodes were added using the “bioanalyzer protocol” (for living single cells and bulk extracted DNA) or the “FFPE protocol” (for fixed single cells), respectively for 17 cycles and 20 cycles of pre-amplication. The quality of purified libraries was verified with the Bioanalyzer (Agilent, USA) and quantified by qPCR using the Ion Library TaqMan™ Quantitation Kit. Libraries were templated using the Ion One-Touch system and sequenced on Ion Torrent™ 318 chips using the Ion Personal Genome Machine™ (ThermoFisher, USA).
Data analysis was performed using the Ion Torrent™ suite (ThermoFisher, USA) and Excel (Microsoft, USA) softwares. Sequencing artifacts in homopolymer regions were filtered out. In theory, when analyzing diploid single cells, only three allele frequencies should be possible: 0% (wild-type), 50% (heterozygous mutant/wild type) and 100% (homozygous mutant). However, WGA and sequencing methods introduce variability and quantitative amplification biases. In consequence, COSMIC hotspot mutations were included in our analysis only if present in at least one single cell WGA sample with a quality score over 100 and 15% allele frequency while using 10X depth amplicon coverage. Venn diagrams were drawn using a public online tool (bioinformatics.psb.ugent.be/webtools/Venn/).