The isolation of fibroblasts and keratinocytes was performed following the protocol of the isolation of various cell lines from corneal tissue.25 (link) Rat skin was stored in keratinocyte medium (Cellsystems, Troisdorf) for up to 4 h after extraction. After removal of the subcutis, the tissue was then cut into pieces of 5 mm which were then incubated in Thermolysin (50 IU/mL) (Sigma-Aldrich) for 15 h at 4°C. The enzyme activity was subsequently stopped with keratinocyte medium. Using sterile forceps (Aeskulap, Tuttlingen), the epidermal layer was then removed and cut into smaller pieces (Figure 3(a) ). These samples were transferred into centrifuge tubes (Greiner Bio-One) filled with TrypLE (Gibco, Waltham) and dissociated by occasional gentle shaking for 30 min at 37°C. Subsequently, the dissociation was stopped with cold keratinocyte medium, and the cell suspension was filtered through a cell strainer (Corning, Corning) and centrifuged. The remaining cell pellet was solved in keratinocyte medium and transferred to cell-culture flasks (Greiner Bio-One) (20 × 103/cm2).
Centrifuge tube
Manufactured by Greiner
Sourced in United Kingdom, Austria
Centrifuge tubes are cylindrical containers used in laboratories to hold samples during centrifugation. They are designed to withstand the high-speed rotation of a centrifuge, which separates components of a sample based on their density differences.
Automatically generated - may contain errors
Lab products found in correlation
Cell strainer, by Corning (1 mentions)
Cell culture flask, by Greiner (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Thermolysin, by Merck Group (1 mentions)
Leucine enkephalin, by Waters Corporation (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase 8, by Merck Group (1 mentions)
Penicillin streptomycin p s, by Lonza (1 mentions)
0.2 m syringe filter, by Avantor (1 mentions)
T75 cell culture flask, by Greiner (1 mentions)
4 protocols using centrifuge tube
1
Isolation of Skin-Derived Cells
2
Analytical Workflow for Biomolecule Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chemicals and materials utilised in this study were: gauze swabs (Arco, UK), sample bags (GE Healthcare WhatmanTM, UK), 15 mL and 50 mL centrifuge tubes (Greiner Bio-One, UK), microcentrifuge tubes 2 mL (Eppendorf, UK), Ministart® 0.2 µm syringe filter (Sartorius, UK), Optima® LC-MS grade solvents 2-propanol, acetonitrile, methanol, and formic acid (Fisher Scientific), HPLC grade HiPerSolv CHROMANORM® ethanol absolute (99.8%), CHROMASOLVTM LC-MS grade water (Honeywell) and Leucine Enkephalin (Waters, Wilmslow, UK).
3
Isolation of Intestinal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Colons and/or caeca were harvested from mice, washed in PBS/BSA and content flushed with forceps. Intestines were then opened longitudinally and washed once more before blotting to remove mucus. Gut tissue was then cut into 1 cm long pieces and placed in a 50 mL centrifuge tube (Greiner) in ice-cold PBS + 0.1% BSA. Colons were incubated two times at 300×g in 40 mL HBSS + 0.1% BSA + 1% Penicillin-Streptomycin (PS, Lonza) + 5 mM EDTA (Sigma-Aldrich) at 37 °C for 10 min before the supernatant was aspirated. Tissue was placed in 40 mL PBS + 0.1% BSA + 1% PS for 5 min. Intestines were then incubated with 20 mL RPMI + 10% FCS + 1% PS + 2.5 U/mL Collagenase VIII (Sigma-Aldrich) + 2 U/mL DNAse I (Roche), shaking for 45 mins–1 h at 37 °C. Supernatant was filtered through a 70-μm-cell strainer to which 30 mL of ice-cold PBS + 0.1% BSA + 1% PS + 5 mM EDTA was added to ablate collagenase/DNase activity. Cells were washed in 30 mL PBS/BSA before filtering once more through a 40-μm-cell strainer. The cells were then pelleted by centrifugation at 400 rcf for 10 min at 4 °C and resuspended in 1 mL RPMI + 10% FCS + 1% PS before counting.
4
Senescent Cell Secretome Modulates MSC Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conditioned medium was generated like the supernatants for the secretome analysis. Briefly, 1.2 × 106 senescent or non-senescent cells were seeded per T75 cell culture flask (Greiner AG, Kremsmünster, Austria). The following day, 20 mL fresh medium was added to each flask and 24 h later, the conditioned medium was transferred to a centrifuge tube (Greiner AG, Kremsmünster, Austria), centrifuged at 522×g for 5 min and the supernatant filtered with 0.2 µm syringe filters (VWR International, Radnor, USA) on SOFT-JECT® syringes (Henke-Sass Wolf, Tuttlingen, Germany). The filtered conditioned medium was divided in aliquots and stored at − 80 °C.
To assess the influence of conditioned medium on the migration of healthy MSCs, the IncuCyte migration tool was used as described above. Therefore, only untreated low-passage MSCs were seeded in the insert plate and regular cell culture medium or conditioned medium was added to the reservoir plate. To reduce the potential problem of nutrient deficiency, also a mixture of 50% conditioned medium with 50% cell culture medium was used. The plates were recorded every 2 h for 188 h in total. The experiment was performed with 8 wells per condition and three times independently.
To assess the influence of conditioned medium on the migration of healthy MSCs, the IncuCyte migration tool was used as described above. Therefore, only untreated low-passage MSCs were seeded in the insert plate and regular cell culture medium or conditioned medium was added to the reservoir plate. To reduce the potential problem of nutrient deficiency, also a mixture of 50% conditioned medium with 50% cell culture medium was used. The plates were recorded every 2 h for 188 h in total. The experiment was performed with 8 wells per condition and three times independently.
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!