Mucosal tissues were rinsed with pre-warmed digestive medium (RPMI1640, anti-fungal-bacterial solution, 2-mM L-Glutamine (all Invitrogen) and 2 mg/ml Collagenase (Sigma-Aldrich, St. Louis)) and minced in 5 ml digestive medium using a scalpel and 19G needle. The minced material was transferred into a 50 ml tube (Greiner) and media was added to 10 ml. Following 20–25 min digestion at 37°C with pulse vortexing every 5 min, samples were transferred into 6-well plates and passed 5 times through a blunt end cannula attached to a syringe. Liberated cells and tissue debris were passed through a 70 µm cell strainer and washed with 30 ml of R10 (RPMI1640 containing anti-fungal-bacterial solution, L-Glutamine and 10% FBS). Cells were resuspended in R10 and equally distributed among FACS tubes. PBMC were isolated using a Ficollpaque (GE healthcare) gradient.
50 ml tube
Manufactured by Greiner
Sourced in Germany
The 50-mL tubes are a type of laboratory equipment used for various applications in research and scientific settings. These tubes provide a standardized container with a capacity of 50 milliliters, allowing for the storage, handling, and processing of samples or reagents.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem and hepes 2.4, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Collagenase, by Merck Group (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Interleukin 2 (il 2), by Novartis (1 mentions)
Rpmi 1640 without phenol red, by Thermo Fisher Scientific (1 mentions)
Luna fl dual fluorescence cell counter, by Logos Biosystems (1 mentions)
K3 edta, by Sarstedt (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Avantor (1 mentions)
Vacutainer system, by BD (1 mentions)
7 protocols using 50 ml tube
1
Isolation of Mucosal Mononuclear Cells
2
Recovering Cryopreserved T Cell Function
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genetically modified T cells display decreased functional activity after cryopreservation [21 (link)]. To recover T cell functions, a resting step according to our previous study [22 (link)] was included, before cellular assays with NY-ESO-1-specific T cells were performed: After thawing and washing, NY-ESO-1-specific T cells were re-suspended at 2 × 106 cells/mL in culture medium supplemented with 600 U/mL IL-2 (Novartis) in a 50 mL tube (Greiner Bio-One, Frickenhausen, Germany). The tube was placed at a slope of 5° above horizon and the cap was unscrewed to allow gas exchange. After an overnight resting period, rested NY-ESO-1-specific T cells were washed, re-counted and used for further experiments.
3
Isolation and Characterization of Human Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were isolated from freshly drawn peripheral blood of healthy donors (NHD). The blood was drawn from a venipuncture into K3 EDTA (Sarstedt AG & Co. KG, Nümbrecht, Germany) tubes and diluted with phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, MA, USA) to a final volume of 35 mL in a 50 mL tube (Greiner bio-one GmbH, Kremsmünster, Austria). The mixture was carefully pipetted on the top of 15 mL Ficoll-Diatrizoate density gradient solution (Lymphoflot, Bio-Rad, Hercules, CA, USA) and centrifuged at 1400 rpm for 30 min at room temperature. PMNs were collected and treated for two brief cycles of hypotonic lysis with ice-cold 36 mL deionized water for 20 s. Osmolarity was restored by addition of 4 mL 10× PBS. Isolated PMNs were resuspended in PBS without calcium and magnesium for counting using the LUNA-FL™ Dual Fluorescence Cell Counter (Logos biosystems, Anyang, Gyeonggi-do, Republic of Korea). The PMN were adjusted to a concentration of 2 million/mL in RPMI-1640 without phenolred (Thermo Fisher Scientific) substituted with 2 mM L-glutamine (Thermo Fisher Scientific) and Penicillin/Streptomycin (Thermo Fisher Scientific) and utilized immediately for the in vitro assays.
4
Thawing and Culturing Frozen PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The frozen PBMCs were thawed in a water bath at 37°C. One milliliter of prewarmed medium (RPMI 1648 medium [Gibco, Darmstadt, Germany] supplemented with 25 mM HEPES buffer [Gibco, Darmstadt, Germany], 1 mM l -glutamine [Gibco, Darmstadt, Germany], 1 × penicillin/streptomycin [PAA; Cölbe, Germany], and 10% heat-inactivated fetal calf serum [FCS; [Gibco, Darmstadt, Germany]) was added to the thawed cells. The cell suspension was transferred to 50-mL tubes (Greiner Bio-One, Frickenhausen, Germany) filled with 8 mL medium (end volume 10 mL). The cells were centrifuged at 400 × g for 6 minutes at room temperature; 1 × 107 cells were resuspended in 10 mL medium and incubated overnight in a cell incubator at 37°C and 5% CO2.
5
Isolation and Stimulation of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density-gradient centrifugation on histopaque 1077 (Sigma), washed three times in Hanks’ balanced salt solution (Life Technologies), and resuspended in a complete medium comprising RPMI 1640 (Sigma) supplemented with penicillin, streptomycin, and L-glutamine (100 U/mL, 100 μg/mL, and 2 mM, respectively) (Sigma).
Participants fasted for 12 h before blood collection (water only, no food intake). Venous blood was drawn into lithium–heparin tubes (BD Vacutainer System). Within 3 h, whole unseparated blood was diluted 1:3 with DMEM and HEPES 2.4% (Invitrogen) and agitated gently in 50 mL tubes (Greiner Bio-one); 1 mL aliquots were seeded in 24-well plates (VWR International) and cultured for 24 h at 37 °C and 5% CO2. From each blood draw, we performed triplicate incubations in parallel with positive and negative controls—separate cultures that each included complete (β5 and β7) and truncated (tβ5 and tβ7) forms. Purified β-conglutin, respectively, LPS and PBS were used for all experiments. Blood cultures were removed from each well and centrifuged at 700× g for 5 min at 20 °C. The supernatants were aliquoted and stored at −20 °C until further analysis.
Participants fasted for 12 h before blood collection (water only, no food intake). Venous blood was drawn into lithium–heparin tubes (BD Vacutainer System). Within 3 h, whole unseparated blood was diluted 1:3 with DMEM and HEPES 2.4% (Invitrogen) and agitated gently in 50 mL tubes (Greiner Bio-one); 1 mL aliquots were seeded in 24-well plates (VWR International) and cultured for 24 h at 37 °C and 5% CO2. From each blood draw, we performed triplicate incubations in parallel with positive and negative controls—separate cultures that each included complete (β5 and β7) and truncated (tβ5 and tβ7) forms. Purified β-conglutin, respectively, LPS and PBS were used for all experiments. Blood cultures were removed from each well and centrifuged at 700× g for 5 min at 20 °C. The supernatants were aliquoted and stored at −20 °C until further analysis.
6
Shockwave Application for Cell Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Shockwaves were applied with a defocused Dermagold 100 device and an OP155 applicator (MTS Medical, Konstanz, Germany). The cells were either stimulated in T25 cell culture flasks, in 15 ml or in 50 ml tubes (Greiner, Kremsmünster, Austria) in PBS with 10% EGM-2. Cells were submerged in a water bath and stimulated with a frequency of 5 Hz, 200 pulses and energy flux densities ranging from 0.03 to 0.19 mJ/mm2 at a constant pressure level of 1 bar as described elsewhere [24] .
7
Blood Culture and PBMC Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Venous blood was drawn into lithium-heparin tubes (BD Vacutainer System, Germany) in the morning. Participants were fasted for 12 hours before blood collection (Fasting consisted in no food or drink intake but water). Within 3 h, whole unseparated blood was diluted 1:3 with Dulbecco's modified Eagle's medium (DMEM) and HEPES 2.4%; (Invitrogen, Germany), and agitated gently in 50mL tubes (Greiner Bio-one, Germany); aliquots (1mL) were seeded in 24-well plates (Nunc, VWR International GmbH, Germany) and cultured for 24 h at 37°C under 5% CO 2 in humidified air. Assays were performed for each blood included positive and negative controls, and separate cultures challenged with 15 µg of each purified βconglutin (β1, β2, β3, β4, β6, respectively). After treatments, blood samples were centrifuged at 700 g for 5 min at 20°C; obtained supernatants were aliquoted and stored at -20°C until further analysis.
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density-gradient centrifugation on Histopaque 1077 (Sigma, USA), washed three times in Hanks' balanced salt solution (Life Technologies, USA), and re-suspended in complete medium consisting of RPMI 1640 (Sigma, USA) supplemented with penicillin, streptomycin and L-glutamine (100 U/mL, 100 µg/mL, and 2 mM, respectively) (Sigma, USA).
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density-gradient centrifugation on Histopaque 1077 (Sigma, USA), washed three times in Hanks' balanced salt solution (Life Technologies, USA), and re-suspended in complete medium consisting of RPMI 1640 (Sigma, USA) supplemented with penicillin, streptomycin and L-glutamine (100 U/mL, 100 µg/mL, and 2 mM, respectively) (Sigma, USA).
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!