Cell-free conditioned medium (CCFM) was prepared from C. pseudodiphtheriticum according to the method of Ramsey et al. (55 (link)) with several modifications. One-liter C. pseudodiphtheriticum cultures were grown for 24 h in BHIT broth at 37°C with shaking at 190 rpm. Cultures were centrifuged at 13,000 rpm for 10 min, and culture supernatant was then passed through a 0.2-μm filter (Corning). Sterile CFCM was further concentrated by ammonium sulfate precipitation. Sterile saturated ammonium sulfate was added to sterile CFCM at 1× the original volume. The suspension was centrifuged at 13,000 rpm for 10 min, and the supernatant was decanted. The pellet was air dried and resuspended in 1× PBS (Fisher Chemicals) at a 50× final concentration. For the disk diffusion assay, S. aureus was cultured in BHI broth overnight at 37°C. The following day, these cultures were diluted to 1 × 108 cells/ml (OD600 of 0.1) in BHI broth, and a sterile swab was used to spread the S. aureus cell suspension on BHIT agar as a lawn. The plate was allowed to dry in a laminar flow hood for 30 min. Next, a sterile 6-mm diffusion disk was place on top of the S. aureus lawn, and 50 μl of CCFM was inoculated onto the disk. Plates were incubated at 28°C, and images were taken after 24 and 72 h of incubation.
0.2 μm filter
Manufactured by Corning
Sourced in United States
The 0.2-μm filter is a laboratory equipment designed for filtration purposes. It features a pore size of 0.2 micrometers, which allows for the separation of particles and microorganisms from solutions or suspensions.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Architect hbsag assay, by Abbott (1 mentions)
Cell scraper, by SPL Life Sciences (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
Synergy htx plate reader, by Agilent Technologies (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ultracentrifugation, by Beckman Coulter (1 mentions)
α mem, by Corning (1 mentions)
L glutamine, by Corning (1 mentions)
Non essential amino acids (neaa), by Corning (1 mentions)
213 ilb, by R&D Systems (1 mentions)
D6046, by Merck Group (1 mentions)
14 protocols using 0.2 μm filter
1
Antibacterial Evaluation of C. pseudodiphtheriticum Conditioned Media
2
Conditioned Medium Preparation from Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 and MCF7 cells were cultured using their culture media until confluence. The cells were then washed with phosphate-buffered saline (PBS) and incubated for 3 days with DMEM/HG supplemented with 100 U/mL P/S or DMEM/F-12 (1:1) supplemented with 2 mM L-glutamine and 100 U/mL P/S for MDA-MB-231 or MCF7 cells, respectively. To prepare conditioned medium (CM) for the control, DMEM/HG supplemented with 100 U/mL P/S or DMEM/F-12 (1:1) supplemented with 2 mM L-glutamine and 100 U/mL P/S was incubated for 3 days under cell-free conditions. The CM from MDA-MB-231 (MDA CM), MCF7 (MCF7 CM), or the control (CON CM) was filtered through a 0.2 μm filter (Corning, Cornyn, NY, USA), aliquoted, and stored at −80 °C until use.
3
HBsAg Quantification from HBV-1.1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conditioned medium from HBV-1.1 cells was harvested, filtered through a 0.2-μm filter (Corning Inc., New York, NY, USA), and used for the Abbott Architect HBsAg assay (Abbott Laboratories, Abbott Park, IL, USA). Results were normalized to cell numbers.
4
Conditioned Media Preparation for Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 and MCF7 cells were cultured in DMEM/high supplemented with 10% FBS and 1% P/S and DMEM/F-12 (1:1) supplemented with 10% FBS and 1% P/S, respectively. After culturing to confluence, the cells were rinsed with PBS and incubated with DMEM/high supplemented with 1% P/S for 3 days. To prepare conditioned medium (CM) for the control, DMEM/high supplemented with 1% P/S was incubated for 3 days under cell-free conditions. Conditioned media from MDA-MB-231 (MDA CM), MCF7 (MCF7 CM), or the control (CON CM) were filtered through a 0.2-μm filter (Corning, NY, USA), aliquoted, and stored at -80 °C before use.
5
Isolation of Human Blood Leukocytes
6
Quorum Sensing Modulation in S. aureus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures of LAC, ΔccpA, ΔcodY, and ΔagrA or each of those strains containing plgt::agrBD were grown to a mid-log OD (~2) in PNG. One mL of cells was removed and pelleted, and the supernatants were filter sterilized through a 0.2 μm filter (Corning). Additionally, overnight cultures of WT LAC harboring the pYFP::RNAIII plasmid (37 (link)) were washed three times with PBS and diluted 1:200 in PNG. Two hundred μL of this culture was added to a 96-well flat-bottom plate (Costar), along with 50 μL of the supernatants prepared earlier. A no supernatant control had an additional 50 μL of PNG added. The plates were grown shaking at 37°C in a Bio-Tek Synergy HTX plate reader overnight with readings being taken every 15 min. Curves were analyzed by highlighting the region of time before the no supernatant control showed YFP expression, and examining activation of the wells with each supernatant added. These experiments were repeated in biological and technical triplicate.
7
Generating Conditioned Media for Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cancer cell lines were cultured until reaching confluence. Subsequently, the media for each cell line were replaced with their respective fresh culture media, containing reduced FBS compared to the complete culture media. Conditioned media for the migration assay were prepared by incubating MDA-MB-231, MCF7, U87, PC3, or LNCaP cells under serum-free conditions for 3 days. BT474, T47D, or SKBR3 cells were cultured with 2% FBS-containing media for 2 days. Conditioned media for the immunostaining of ectopic ZO-1 expression were prepared by incubating MDA-MB-231 cells with 2% FBS containing culture media for 2 days. To prepare conditioned media for the control, FBS-deprived or FBS-reduced media specific to each cell line were incubated for the same period under cell-free condition. The conditioned media were filtered through a 0.2 μm filter (Corning, Corning, NY, USA), aliquoted, and stored at −80 °C until use.
8
Isolation of Tumor-Derived Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate tumor‐derived extracellular vesicles (EVs) from cultures, cells were grown in T175 cm2 flasks until 70–80% confluence for 72 h in 30 mL of FBS‐depleted media (in the case of tumorspheres cultures). After 72 h, detritus was eliminated by differential centrifugation at 500 g for 5 min, and then at 3000 g for 15 min. Subsequently, the supernatant was filtered through a 0.2‐μm filter (Corning, NY, USA) and ultracentrifuged at 110 000 g for 90 min (CP‐NX, P50AT2 Rotor; Hitachi, Japan). To wash the first pellet, second ultracentrifugation was performed; EVs were then resuspended in 30 mL of phosphate‐buffered saline (PBS). All centrifugations were performed at 4 °C. At last, EVs were resuspended in a tiny volume (30–60 μL) of filtered PBS and stored at −80 °C until the corresponding analysis.
9
Optimizing hPL Supplementation for hAF-MSC Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pooled human platelet concentrate (hPC) was obtained by pooling 15 platelet apheresis collections from the Laboratory of Blood Bank Section, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University. All pooled hPC underwent three cycles of being frozen and then thawed at −80 °C and 37 °C, respectively. The remaining cellular fragments were eliminated by centrifugation at 4000 rpm for 20 min. After that, the supernatant was filtered using a 0.2 μm filter (Corning Incorporated, NY, USA) and 4U/ml of heparin was added to prevent gel formation. The final stock of hPL was kept at −20 °C.
To determine the impact of utilizing different serum types on cell viability and to evaluate the optimal concentration of hPL that was used as a supplement in the basal growth medium, the isolated hAF-MSCs were assessed using two different assays: Methylthiazol tetrazolium (MTT) and Alamar blue proliferation analysis.
To determine the impact of utilizing different serum types on cell viability and to evaluate the optimal concentration of hPL that was used as a supplement in the basal growth medium, the isolated hAF-MSCs were assessed using two different assays: Methylthiazol tetrazolium (MTT) and Alamar blue proliferation analysis.
10
Immunoblotting of GAS Virulence Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Overnight cultures were inoculated into fresh culture medium and grown to late-exponential phase (OD600 of 0.8). Bacterial cultures were centrifuged at 11,000 × g for 3 min at room temperature, and supernatants were passed through a 0.2-μm filter (Corning, USA). GAS pellets or supernatants were mixed with 2 × sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled at 100°C for 10 min, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (0.45 μm; Merck Millipore). Nga and SLO bands were visualized by standard immunoblotting and chemiluminescence methods.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!