0.22 mm filter

Manufactured by Merck Group

Sourced in United States, Germany

The 0.22-mm filter is a laboratory equipment used for filtration. It is designed to remove particles, microorganisms, and other contaminants from liquids and gas samples. The filter has a pore size of 0.22 millimeters, which allows the passage of the desired substance while retaining unwanted materials.

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Lab products found in correlation

78 protocols using 0.22 mm filter

Generating CRISPR Knockout Cell Lines

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The lentiCRISPRv2 plasmid was a gift of Professor Shi Liu (Wuhan University). A specific oligo targeting the gene was designed using the Cas9 target design tools (http://www.genome-engineering.org). The designed oligos are listed in Additional file 1: Table S3. The target guide sequence cloning protocol can be found at the Zhang Laboratory GeCKO Web site (http://www.genome-engineering.org/gecko/). The specific lentiCRISPRv2 plasmid, lentivirus packaging plasmid psPAX2, and envelope plasmid pMD2.G were cotransfected into 293 T cells in 60-mm culture dishes using Lipofectamine 3000. The harvested medium was centrifuged at 15,000×g for 5 min and then filtered through a 0.22-mm filter (Millipore) to remove cells. When cells were grown to ∼70% confluence, they were incubated in fresh culture medium containing 8 mg/ml polybrene. Subsequently, we added specific lentiCRISPRv2 lentivirus-containing media to the cells. The monoclonal cells were singled out for enlarged culture. KO cell lines were obtained from these enlarged monoclonal cells, and KO cells were confirmed by qRT-PCR and western blotting (Additional file 1: Fig. S1B).

Li W., Li F., Lei W, & Tao Z. (2019). TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination. Cell Communication and Signaling : CCS, 17, 162.

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Retroviral Packaging and Transduction Protocol

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5×10^6 SD3443 retroviral packaging cells were plated per 100mm dish overnight in DMEM complete. After 24 hours, media was replaced with 5 ml serum-free DMEM and plates were treated with 25 uM chloroquine for at least 15 minutes. Cells were transfected with 4 μg of DNA using Lipofectamine Plus transfection reagent (Invitrogen) per manufacturer’s protocol for retrovirus packaging. After 4 hours, 5 ml complete DMEM with 20% FBS was added to the plate. The next day, the media was replaced with 5 mls fresh DMEM complete with 10% FBS. After 24 hours, retrovirus was collected by harvesting the supernatant and filtering it through a 0.22 mm filter (Millipore, Billarica, MA) to remove cell particulates and ensure sterility. Retrovirus was either used fresh or stored at −80°C until needed.
To transduce cells, 1.5 ml retroviral stock supplemented with 1.5 μl polybrene was added to cells in T75 flasks at 40–60% confluency. Cells were incubated with the retrovirus on a gentle rocker at 37°C. After 2 hours, the retrovirus was removed and replaced with media appropriate to the cell type. Cells were allowed to grow to approximately 80%, which occurred within 1–3 days. For cell selection, geneticin (G418) (Invitrogen) at a concentration of 75–100 μg/ml was used and selection was maintained until all the cells in the control (uninfected) flask died.

Sudarshan S.R., Schlegel R, & Liu X. (2022). Two conserved amino acids differentiate the biology of high‐risk and low‐risk HPV E5 proteins. Journal of Medical Virology, 94(9), 4565-4575.

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Assessing Celecoxib's Impact on PGE2 Levels

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The ovarian cancer cell lines were plated in 24 well plates and cultured for 24 hours. The cells were then treated with celecoxib for 18 hours. The culture media was harvested using a syringe and filtered through a 0.22-mm filter (Millipore, Billerica, MA). PGE2 productions in the media was determined by quantitative enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's instructions. PGE2 concentration was measured in triplicate for each cell line. The concentration of PGE2 in the serum of mice treated with celecoxib was also assessed by this technique.

Suri A., Sheng X., Schuler K.M., Zhong Y., Han X., Jones H.M., Gehrig P.A., Zhou C, & Bae-Jump V.L. (2016). The effect of celecoxib on tumor growth in ovarian cancer cells and a genetically engineered mouse model of serous ovarian cancer. Oncotarget, 7(26), 39582-39594.

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Exosome Isolation from Prostatic Fluid

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The prostatic fluid sample from each individual was collected in a 1.5-mL sterile Eppendorf tube as described in the literature (25 (link)), followed by ultracentrifugation at 3,000 g and 4 °C for 10 min to collect the supernatant and a dilution with an equal volume of sterile phosphate-buffered saline (PBS, pH 7.4). The diluted supernatant was further centrifuged at 10,000 g and 4 °C for 30 min, followed by collecting and filtering the supernatant with a 0.22-mm filter (Millipore, Burlington, MA). The filtered supernatant was further centrifuged at 150,000 g and 4 °C for 2 h in a CP70ME ultracentrifuge (Hitachi, Japan). To further purify the exosomes, the pellet was washed with a large amount of sterile PBS, centrifuged at 150,000 g and 4 °C for 1 h to collect the precipitate, and resuspended in 100 µL sterile PBS for subsequent experiments.

Zhao B., Zheng J., Qiao Y., Wang Y., Luo Y., Zhang D., Cai Q., Xu Y., Zhou Z, & Shen W. (2021). Prostatic fluid exosome-mediated microRNA-155 promotes the pathogenesis of type IIIA chronic prostatitis. Translational Andrology and Urology, 10(5), 1976-1987.

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Exosome Isolation from Huh7 Cell Culture

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The culture medium of Huh7 cells was centrifuged at 3000 g for 15 min, and a 0.22 mm filter (Millipore) was used to filter the supernatant. The culture medium was then mixed with Exoquick exosome precipitation solution (System Biosciences, Beijing, China) for 24 hours. After centrifuging at 1500g for 30 minutes, the supernatant was discarded. Exosomes were suspended in phosphate buffered saline (PBS) solution for the following experiments. Exosomes were identified by transmission electron microscopy (TEM) and Western blot assay.

Wang D., Xing N., Yang T., Liu J., Zhao H., He J., Ai Y, & Yang J. (2020). Exosomal lncRNA H19 promotes the progression of hepatocellular carcinoma treated with Propofol via miR‐520a‐3p/LIMK1 axis. Cancer Medicine, 9(19), 7218-7230.

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CRISPR-mediated Gene Knockdown in HEK293 and THP1 Cells

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Double-stranded oligonucleotides corresponding to the target sequences were cloned into the lenti-CRISPR-V2 vector and co-transfected packaging plasmids into HEK293 cells. 48 h after transfection, the viruses were harvested, ultra-filtrated (0.22 mm filter, Millipore) and used to infect HEK293T or THP1 cells in the presence of polybrene (8 μg/mL). The infected cells were selected with puromycin (2 μg/mL) for at least 5 days. Human HDAC3 and TBK1 sgRNA targeting sequences were in Table S4.

Tang J.L., Yang Q., Xu C.H., Zhao H., Liu Y.L., Liu C.Y., Zhou Y., Gai D.W., Pei R.J., Wang Y., Hu X., Zhong B., Wang Y.Y., Chen X.W, & Chen J.Z. (2020). Histone deacetylase 3 promotes innate antiviral immunity through deacetylation of TBK1. Protein & Cell, 12(4), 261-278.

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MTT Assay for Cell Viability

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This protocol was used for the evaluation of cell viability and all the cell lines. It must be noted that this assay describes the count of cells that remain alive after the treatment. For the experimental procedure a standard solution of MTT 1mg/mL in PBS was prepared which after filtration (0.22 mm filter (Millipore, Carrigtwohill, Ireland)) was stored in the dark (4–8 °C) until use. In a 96-well plate, HaCaT, A549 and A375 cells were seeded in a number of 30 × 10⁴ cells/well for each evaluation series respectively. The cells were incubated (37 °C, 5% CO₂) for 24 h, followed by the addition of the tested analogues (100 μM, diluted in DMEM) and another 24-h incubation. Next, appropriate amount of MTT was added in the tested wells and the plate returned to the incubator for the next 4 h for the formation of the formazan crystals. The formazan crystals were then redissolved by adding DMSO (100 μL) and stirring lightly for 40 min, after which the absorbance was measured in a plate reader at 540 nm with a reference absorbance of 720 nm.
All samples were tested in triplicate, against a control (only cells), blank (no cells), and positive control (100 μM standard silibinin solution was used as a “cell-killing” standard).

Katopodi A., Tsotsou E., Iliou T., Deligiannidou G.E., Pontiki E., Kontogiorgis C., Tsopelas F, & Detsi A. (2021). Synthesis, Bioactivity, Pharmacokinetic and Biomimetic Properties of Multi-Substituted Coumarin Derivatives. Molecules, 26(19), 5999.

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Bacterial Supernatant Concentration Protocol

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Bacterial inoculum was prepared as described above. Subsequently, 1mL of the culture with OD_600nm of 0.1 was inoculated into 1000mL of LB broth and grown to stationary phase for 20h. The culture was centrifuged at 20000xg for 40mins at 4°C and the resulting supernatant was filtered through a 0.22 mm filter (Millipore, USA) to obtain bacteria-free culture supernatant, and concentrated using ultra-filtration as described [20 (link)], with minor modifications. Briefly, the culture supernatant was concentrated 20-fold using a Quixstand bench top system (GE Healthcare, Darmstadt, Germany). The supernatant obtained was further concentrated to 50-fold by ultra-filtration employing 10kDa centricon ultra-free centrifugal filter units (Millipore, Massachusetts, USA). The samples were subjected to overnight dialysis using 0.1M phosphate buffered saline (PBS) and the protein concentration was determined using the Bradford method [18 (link)].

Vellasamy K.M., Mariappan V., Shankar E.M, & Vadivelu J. (2016). Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells. PLoS Neglected Tropical Diseases, 10(7), e0004730.

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Isolation and Characterization of A549-Derived Exosomes

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The fresh complete medium with EV-free FBS (Thermo, America) was used to incubate A549 cells for 48 h. Thereafter, the cells underwent centrifugation at 1500 g and 4 °C for 15 min. After filtration by a 0.22 mm filter (Millipore, America), the supernatant underwent two sequential centrifugations at a high speed of 110,000 g and a temperature of 4 °C for 1 h. A549-Exos were then resuspended in an appropriate volume of phosphate-buffered saline (PBS) and kept at –80 °C for subsequent application. The morphological feature of A549-Exos was observed using transmission electron microscopy. A Nano-z90 Nanosizer (Malvern, UK) was allied to evaluate the size distribution of A549-Exos. Western blot method was allied to analyze the expressional levels of exosomal surface markers such as CD9 and TSG101.

Zhang C., Qin J., Yang L., Zhu Z., Yang J., Su W., Deng H, & Wang Z. (2022). Exosomal miR-328 originated from pulmonary adenocarcinoma cells enhances osteoclastogenesis via downregulating Nrp-2 expression. Cell Death Discovery, 8, 405.

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Isolation and Characterization of Extracellular Vesicles from Hypoxic and Normoxic hADSCs

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hADSCs at passage 3 were cultured under hypoxia (5% oxygen) and normoxia (20% oxygen) until cells reached 70–80% confluency. The medium was then replaced with serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 for 24 h to collect conditioned medium. Cell debris and apoptotic bodies were discarded after sequential centrifugations at 500 ×g, 3,000 ×g and 10,000 ×g for 5, 15 and 60 min, respectively. The supernatant was then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h using a 45 Ti rotor (Beckman Coulter, Optimal L-80XP, USA). The remaining precipitate was resuspended in PBS and filtered with a 0.22-mm filter (Millipore, Billerica, MA, USA), then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h. EVs were stored at −80 °C until further use. EVs obtained from both conditions were characterised using 80 kV TEM (HITACHI H-7000FA, Japan). The particle size distribution and concentrations of EVs were analysed by ZetaVIEW (Particle Metrix, ZetaVIEW S/N 17–315, Germany) and Nanoparticle Tracking Analysis software (ZetaVIEW 8.04.02). Antibodies against CD9, CD63, Alix, β-actin and calnexin (Abcam, London, UK) were used in western blotting. In addition, the supernatant of EV lysates was prepared, and the protein content was evaluated using a BCA Protein Assay Kit (Sigma, Silicon Valley, USA).

Zhu Y., Zhang J., Hu X., Wang Z., Wu S, & Yi Y. (2020). Extracellular vesicles derived from human adipose-derived stem cells promote the exogenous angiogenesis of fat grafts via the let-7/AGO1/VEGF signalling pathway. Scientific Reports, 10, 5313.

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