Ten eyes of 2.5-month-old Krt1–15-EGFP mice were enucleated, the limbus (with marginal conjunctiva and peripheral cornea) was dissected (~0.5mm), tissues were pooled and incubated in 300μl of trypsin (X10 Biological Industries) for 10 minutes (37°C). Supernatant was collected into 10ml RPMI (Biological Industries) containing 10% chelated fetal calf serum. Trypsinization was repeated for 10 cycles adding fresh trypsin in each cycle. Cell suspension was centrifuged (8 minutes at 300g), re-suspended, and filtered using a cell strainer (VWR) to achieve 1200cells/μL
Cell strainer
Manufactured by Avantor
Sourced in United States, Germany
A cell strainer is a laboratory device used to filter and separate cells from a liquid suspension. It consists of a fine mesh screen that allows the passage of cells and small particles while retaining larger cell aggregates, debris, or clumps.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Yatalase, by Takara Bio (2 mentions)
Glucanex, by Merck Group (2 mentions)
Roswell park memorial institute (rpmi), by Sartorius (1 mentions)
C1 single cell autoprep ifc microfluidic chip, by Standard BioTools (1 mentions)
C1 instrument, by Standard BioTools (1 mentions)
Te2000 e, by Nikon (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Sybr green pcr mix, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127 gel, by Merck Group (1 mentions)
Mouse elisa kit, by R&D Systems (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Collagenase 5, by Merck Group (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
15 protocols using cell strainer
1
Isolation of Murine Limbal Progenitor Cells
2
Single-Cell RNA-seq of Neural Stem Cells
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Sai2 NES cells passage 29, AF22 NES cells passage 20 and Ctrl‐7 NES cells passage 12 were harvested as neural stem cells at day 0 and Sai2 DIFF passage 27, AF22 DIFF passage 16 and 23, Ctrl‐7 DIFF passage 12 as differentiated neurons at day 28 of differentiation.
The cells were dissociated as described under neural stem cell culture. The dissociated cells were passaged through a cell strainer (40 μm, VWR) and diluted to 2,000 cells/ml in NES medium with addition of 5% DNaseI (2,000 U/ml, Qiagen) and 1% BSA (Sigma). Cells were placed on ice until further processed. The cells were loaded according to manufacturers protocol on C1 Single‐Cell AutoPrep IFC microfluidic chip (for cell size 10–17 μm) and processed on a Fluidigm C1 instrument. This chip contains 96 wells for single‐cell capture.
Each chamber of the chip was optically inspected by automated microscope (Nikon TE2000E), empty chambers, cell clumps and dying cells were discarded from further analyze. Lysis of the cells and cDNA production were carried out according to the Linnarsson's laboratory protocol (Islam et al.,2014 ). For analysis, high‐quality single cells were selected through cutoff to fit between 500 and 5,000 genes/cell.
The cells were dissociated as described under neural stem cell culture. The dissociated cells were passaged through a cell strainer (40 μm, VWR) and diluted to 2,000 cells/ml in NES medium with addition of 5% DNaseI (2,000 U/ml, Qiagen) and 1% BSA (Sigma). Cells were placed on ice until further processed. The cells were loaded according to manufacturers protocol on C1 Single‐Cell AutoPrep IFC microfluidic chip (for cell size 10–17 μm) and processed on a Fluidigm C1 instrument. This chip contains 96 wells for single‐cell capture.
Each chamber of the chip was optically inspected by automated microscope (Nikon TE2000E), empty chambers, cell clumps and dying cells were discarded from further analyze. Lysis of the cells and cDNA production were carried out according to the Linnarsson's laboratory protocol (Islam et al.,
3
Single-cell Cytokine Analysis of Virus-infected Splenocytes
4
Adenoviral CCL28 Expression Protocol
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F-127 Pluronic gel, DTT, BSA, and RIPA buffer were from Sigma (St. Louis, MO). n-Octyl-β-d -glucopyranoside (ODG) was from RPI Corp (Mt Prospect, IL). Mouse neutralizing anti-CCL28 antibody and mouse ELISA kits were purchased from R&D Systems (Minneapolis, MN). Mouse anti-CCR10 and mouse anti-eNOS antibodies were from Santa Cruz Biotechnology (Dallas, TX). 4′,6-Diamidino-2-phenylindole (DAPI), fluorescently labeled secondary antibodies, Trizol and SYBR Green PCR mix were from ThermoFisher Scientific (Waltham, MA). Mouse anti-eNOS and mouse anti-actin antibodies were from BD Biosciences (San Diego, CA). Rabbit monoclonal anti-CD31 and Griess Reagent kit were from Cell Signaling Technology (Danvers, MA). Nitrocellulose membrane was from Bio-Rad Laboratories (Hercules, CA). SuperSignal West Femto Kit and Restore Western Stripping buffer were from Pierce (Rockford, IL). Skin punch biopsy tool was from Acuderm Inc. (Fort Lauderdale, FL). Cell strainer was from VWR Scientific (Franklin, MA). Control and CCL28 adenoviral expression vectors (Adv-Ctl, Adv-CCL28) were generated by Dr. Jody L. Martin (UIC Vector Core and Department of Pharmacology, University of California, Davis).
5
Isolation and Characterization of Murine BMSCs
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BMSCs were obtained from the bone marrow of femurs and tibias of APN KO or C57BL/6J (B6) mice. At least 3-to 4-month-old APN KO or B6 mice were used for young BMSCs, and 11 to 12-month-old APN KO or B6 mice were used for old BMSCs. Specifically, whole bone marrow from the femur and tibia was flushed with 1X phosphate-buffered saline (PBS) with penicillin–streptomycin (Sigma) and passed through a 70 µm of cell strainer (VWR), and 1X RBCs lysis buffer (BioLegend) was used to remove erythrocytes from the BM. Cells were plated in Mesencult™ Media with 1% L-Glutamine (Sigma) and penicillin–streptomycin (Sigma), incubated in a 10% CO2, 1% O2 hypoxia chamber until 80% confluence, then cells were sub-cultured 1:2 with half media, and all experiments were performed using cells at passage two or three. Moreover, the phenotype of BMSCs was characterized using a flow cytometry cell detachment solution (BioLegend).
6
Trichome Isolation and Enrichment
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Trichome release and enrichment were carried out according to the STIRRER method introduced previously (Huebbers et al. 2022 (link)). Briefly, freshly harvested A. thaliana seedlings were transferred to a 500-mL beaker containing 250 mL of phosphate-buffered saline solution (PBS; 10-mM disodium hydrogen phosphate, 2.7-mM potassium chloride, 2.0-mM potassium dihydrogen phosphate, 0.5-mM magnesium chloride, 137-mM sodium chloride, pH 7.5) supplemented with 50-mM ethylenediaminetetraacetic acid (EDTA). The trichome buffer suspension was stirred at 300 rpm for 30 min. Subsequently, 4 layers of screen door mesh (nominal pore size of 1.2 × 1.4 mm) were used to separate the processed seedlings from the buffer solution containing the released trichomes. The trichomes were captured in a 300-mL beaker and poured through a cell strainer (VWR, Radnor, Pennsylvania, USA) with a nominal pore size of 100 µm. These strained trichomes were collected by a small spatula and transferred to 2-mL reaction tubes. After the first round of agitation and filtration, the process was repeated 2 times for 15 min each. Enriched trichomes were either stored at 4 °C in 1-mL phosphate-buffered saline buffer or frozen in liquid nitrogen and lyophilized for monosaccharide and cellulose quantification or FTIR spectroscopy.
7
Isolation of Intestinal and Lymphoid Cells
8
Isolation and sequencing of iMPCs
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A transgene-free stable iMPC clone at passage 6 was collected by trypsinization from a cell culture plate. To remove cell debris and multi-nucleated myotubes present in iMPCs culture, collected cells were filtered through a 40 µm cell strainer (VWR, Cat. #734-0002). Filtered cells were then resuspended in PBS and used for cell counting. Cell viability was checked with Trypan blue (Sigma-Aldrich, Cat. #T8154) staining. Next, cells were diluted in PBS at 1000 cells/µl and used by a 10x Genomics platform. Chromium Next GEM Single cell 3’ v3.1 protocol was used according to the manufacturer’s instruction. In short, a Gel Bead-In Emulsions (GEM) was generated by loading cells in chromium Next GEM chip G targeting ~5000 cells in recovery. GEM was then incubated in a thermal cycler and cleaned with Dynabeads followed by cDNA amplification. Amplified cDNA was used for fragmentation followed by end repair and A-tailing. Next, adaptor ligation and index PCR were performed using single index plate T set A and double size selection was carried out using AMPure XP (Beckman Coulter, Cat. #A63881). The library was sequenced on Novaseq 6000 (Illumina, Inc, California, USA) with paired ends.
9
Activated Sludge Cell Isolation Protocol
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Activated sludge (30 mL) from aeration tanks was obtained at the AAW WWTP on 16 December 2020 and 03 March 2021 and immediately homogenized in the lab using the RZR 2020 Benchtop Stirrer (Heidolph, Schwabach, Germany) with a glass/Teflon tissue grinder attached (1 min, 2nd gear). Five milliliters was subsequently sonicated using a Bandelin Sonopuls HD2200 with an MS73 probe (Berlin, Germany) set at 60% amplitude with 6 × 10 s pulses with a 10-s interval. Single cells were separated by centrifugation through a cell strainer with pore sizes of 40 µm (VWR) for 5 min at 3,000 × g, followed by centrifugation through a cell strainer with pore sizes of 5 µm (pluriSelect Life Science, Leipzig, Germany) for 2 min at 8,600 × g. Cells present in the permeate were counted in a Bürker-Türk counting chamber and subsequently diluted to approx. 10,000 cells/mL.
10
Isolation of Murine Lung Cells
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Dissected lungs were placed in RPMI buffer (RPMI Glutamax [Gibco, Amarillo, TX, USA] + 5% fetal calf serum (FCS) + 1% penicillin/streptomycin + 0.01% β-mercaptoethanol) and were cut to small pieces with scissors. Next, digestion medium (RPMI buffer + 2 mg/ml collagenase D + 0.1 mg/ml DNase I) was added to the lungs prior to an incubation step of 30 min at 37°C. Lung tissue was further homogenized with a syringe and needle and digestion medium was added for another incubation step of 15 min at 37°C. Next, tissue was mechanically homogenized for a second time with syringe and needle. After a centrifugation step (5 min, 708 g, RT), cells were resuspended in 10 mM EDTA and PBS + 2% FCS. Cells were centrifuged (5 min, 708 g, RT) and treated with NH4Cl buffer for 3 min at 37°C to lyse RBCs. After passage through a 70 μm cell strainer (VWR) cells were resuspended in PBS + 2% FCS and live cells were counted upon staining with trypan blue.
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