The in vitro cytocompatibility/cytotoxicity tests were developed on two types of healthy cell lines and on two tumor cell lines: immortalized human keratinocytes (HaCaT - 300493; CLS Cell Lines Service GmbH), primary epidermal melanocytes (HEMa - ATCC® PCS-200-013™), human melanoma (A375 - ATCC® CRL-1619™), and murine melanoma (B164A5 – 94042254; ECACC). HaCaT, A375, and B164A5 cells were cultured in specific culture medium - Dulbecco’s modified Eagle Medium high glucose supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution. HEMa cells culture required Dermal Cell Basal Medium supplemented with Adult Melanocyte growth kit, 1% penicillin/streptomycin mixture, and 1% FCS. Throughout the experiments, the cells were maintained in a humidified incubator in standard conditions (5% CO2 at 37°C) and were passaged every other day. The cells were counted using Countess™ II Automated Cell Counter in the presence of Trypan blue.
Countess 2 automated cell counter
Manufactured by Countstar
The Countess® II Automated Cell Counter is a laboratory instrument designed to quickly and accurately count cells. It uses advanced imaging technology to analyze samples and provide cell counts and viability information.
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10 protocols using countess 2 automated cell counter
1
In Vitro Cytocompatibility and Cytotoxicity Tests
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Rice Protoplast Isolation and Enumeration
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The rice protoplast was isolated using the previous methods (Jabnoune et al., 2015 (link); Wang et al., 2021 (link)). Briefly, the finely cut rice seedling leaf sheaths were immediately incubated in an enzymes solution containing 10 mM MES, 0.6 M mannitol, 0.75% macerozyme R-10, and 1.5% cellulase RS (pH 5.7) for 3 h (shaking at 70 rpm) at 28°C. After incubation, the enzyme solution was filtered out. The digested protoplasts were washed with the W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES at pH 5.7) and collected by centrifugation at 300 × g. The protoplasts were resuspended in a solution containing 0.6 M D-Mannitol, 15 mM MgCl2, and 4 mM MES (pH 5.7). A small amount of the single-cell suspension was added to an equal volume of 0.4% trypan blue dye. The concentration of viable cells was adjusted to the desired concentration (1000 to 2000 cells/µL) by counting the cells using Countess® II Automated Cell Counter.
3
Isolation of PBMCs from Whole Blood
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Blood diluted twofold in Roswell Park Memorial Institute (RPMI) 1640 (with l -glutamine and penicillin–streptomycin) was gently overlayed on Ficoll-Paque™ under sterile conditions and centrifuged at room temperature for 10 minutes at 400×g without breaks. The top plasma layer was carefully pipetted away, and mononuclear cells were collected and washed twice with RPMI–5% fetal bovine serum (FBS). PBMCs were counted using the Countess II automated cell counter and cryopreserved in liquid nitrogen at 20 million cells per mL in a cryo-vial until further analyses.
4
MCF-7 Cell Viability Assay
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MCF-7 cells were seeded at a confluence of 50.000 cells/well in 24-well plates and treated for 48 h with CM. Cells were harvested by trypsinization and incubated in a 0.5% trypan blue solution for 10 min at room temperature. Trypan blue negative cells were counted through a Countess® II Automated Cell Counter at day 0 and after 48 h of treatment.
5
Culturing Pancreatic Cancer and CAF Cells
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Pancreatic cancer cell line (ASPC-1) and CAF cells (ATCC #CRL-1682, ATCC #PCS-201-012) were obtained from the American Type Culture Collection (ATCC). ASPC-1 cells were chosen based on their metastatic traits and to model PDAC allowing us to observe the effects on the EMT processes [28 (link)]. Cells were cultured in RPMI, and DMEM High glucose media, respectively. All cell culture media were prepared by fetal bovine serum (FBS) with a final concentration of 10% (v/v) and Penicillin-Streptomycin (50 mg/mL streptomycin in 0.9% NaCl and 50,000 U/mL penicillin in 0.9% NaCl) with a final concentration of 1% (v/v). Cells were thawed in a 37 °C water bath and added to a tube with 9 ml of warm media. The suspension was centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in fresh media and transferred to a clean cell culture flask. Cells were cultured until reaching 80% confluency and removed from the flask with the help of Trypsin (Trypsin-EDTA (0.05%), phenol red), and the trypsin activity was stopped by adding fresh culture media containing FBS. The cells were centrifuged, resuspended, and then counted using Countess II Automated Cell Counter for downstream experiments.
6
Single-cell RNA-seq Quality Control
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The single-cell suspension was generated at 700–1200/ul (viability ≥85%) as determined using the Countess® II Automated Cell Counter. Raw data (Raw Reads) of FASTQ files were transformed from the Raw BCL files using Illumina’s bcl2fastq converter and for Raw data, firstly processed through primary quality control. Reads were removed out of the downstream analysis when they meet one of the following three conditions: (1) contain N base more than 3; (2) with more than 20% bases with Phred <5; (3) have adapter sequences. All the downstream analyses were based on clean data with high quality.
7
Isolation of Single Cells from Deep Fascia
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A sterile RNase-free culture dish was placed on ice with PBS. The deep fascia samples were obtained from the medial shank of all participants. We cleaned blood spots and adipose tissue with 1×PBS and trimmed them into 0.5 mm2 fragments. Tissues were dissociated into single cells in a 37°C water bath with shaking at 100 rpm for 20 minutes in a dissociation solution (0.35% collagenase IV5, 2 mg/ml papain, 120 units/ml DNase I). Decomposition was stopped by pipetting 5-10 times with a Pasteur pipette in 1× PBS containing 10% fetal bovine serum (FBS, V/V). The cell suspension was then filtered using a 70-30 µm stacked cell strainer before being centrifuged at 300 × g for 5 minutes at 4°C. Single-cell suspensions were counted using a hemocytometer/Countess II Automated Cell Counter, and the concentration was adjusted to 700-1200 cells/μl. Overall cell viability was validated by trypan blue exclusion, which was required to be above 85%.
8
Single-cell sequencing library construction
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Samples were prepared into a single‐cell suspension and examined for cell count and cell viability using a Countess® II Automated Cell Counter. Single‐cell suspensions with cell activity above 80% and a cell concentration of 1000 cells/μl were mixed with 10× Barcode Gel Beads and enzyme to construct a 10× Genomics labelled single‐cell library in accordance with the manufacturer's instructions. The Illumina HiSeq platform was used for sequencing of the library. Raw reads were aligned to the reference genome using STAR cell ranger, and unique alignment sequences were selected for subsequent analysis. The Unique Molecular Identifier (UMI) was calibrated based on the unique RNA sequence alignment results. After removal of duplicates, UMI counting was carried out for the different genes for each Barcode to determine the effective cells.
9
Single-Cell Testis Dissociation for scRNA-seq
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The testis samples from each individual were cut into smaller pieces with the volume less than 0.5 mm3 after washing three times with PBS, then subjected to enzymatic digestion comprising Collagenase IV (1 mg/mL) and Dnase I (1 μg/μL) in a 1.5 mL centrifuge tube at 37 °C for 15 min as described previously [16 (link), 26 (link)]. During the digestion period, the tissues were mixed by pipetting up and down once in every five minutes. Enzymic digestion was terminated by adding 10% FBS (in DMEM) and testicular single cells were obtained by filtering through a 40-μm nylon mesh. After centrifugation at 1500 rpm for 5 min at 4 °C, the supernatant material was removed and the single cells were washed with 0.04% BSA DPBS for three times and the cell number and cell viability were measured by using the Countess® II Automated Cell Counter. The cells were resuscitated to a concentration of 700–1200 cells/μL with a viability ≥ 85%, ready for scRNA-seq.
10
A375 Melanoma Cell Culture
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A375 melanoma cells require the following cell growth conditions: a specific culture medium-Dulbecco's Modified Eagle's Medium (DMEM-ATCC® 30-2002™) high glucose-4500 mg/L enriched with 10% fetal bovine serum (FBS) and 1% mixture of antibiotics (Penicillin and Streptomycin) to avoid cells' contamination. Through the experiments, the cells were incubated in standard conditions (at 37 • C and 5% CO 2 ) and cultured according to the manufacturers' recommendations. The cells were counted automatically by the means of Countess™ II Automated Cell Counter and Trypan blue.
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