Testicular cell suspensions were prepared by a procedure previously described in our laboratory [27 , 29 (link)]. Reagents and plasticware were handled in RNAse-free conditions. Briefly, testes were dissected into 35 mm glass Petri dishes containing ice-cold separation medium (10 % v/v fetal calf serum in Dulbecco’s Modified Eagle’s medium [DMEM], with high glucose and L-glutamine), and cut into 8–10 mm3 pieces after removal of the tunica albuginea. Between three and four of these pieces per round were immediately placed into a sterile disposable disaggregator unit (Medicon; Beckton-Dickinson [BD], CA) plus 1 mL of ice-cold separation medium, and processed for 30 sec in the Medimachine System (BD), an automated electro-mechanical solid-tissue disaggregator. The resulting cell suspension was recovered from the Medicon unit with a 5 mL disposable syringe, sequentially passed through two 50 μm Filcon filter units (BD), and placed on ice. Cells were counted by means of a Neubauer chamber and resuspended at a concentration of up to 3–5 × 106 cells/mL in separation medium. NDA (2-naphthol-6,8-disulfonic acid, dipotassium salt; Chemos GmbH, Regenstauf, Germany) was added to the suspension to a final concentration of 0.2 % (w/v) in order to prevent cell clumping.
Medicon
Manufactured by BD
Sourced in United States
Medicon is a versatile laboratory equipment designed for fundamental laboratory processes. It offers reliable performance and consistent results for a variety of essential tasks in the scientific and medical research fields.
Automatically generated - may contain errors
Lab products found in correlation
Medimachine, by BD (2 mentions)
Medimachine system, by BD (2 mentions)
Histopaque 1119, by Merck Group (2 mentions)
Histopaque, by Merck Group (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Gr1 ly6c, by BD (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Cd11b, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Epics xl mcl flow cytometer, by Beckman Coulter (1 mentions)
Filcon, by BD (1 mentions)
Sc 17773, by Santa Cruz Biotechnology (1 mentions)
Versene edta solution, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Anti cd14 apc, by Thermo Fisher Scientific (1 mentions)
Elisa, by R&D Systems (1 mentions)
Dnase, by Merck Group (1 mentions)
8 protocols using medicon
1
Isolation of Testicular Cell Suspensions
2
Isolation of Liver-Infiltrating Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of liver-infiltrating lymphocytes was performed by an automated, mechanical disaggregation system (Medimachine, Becton Dickinson, USA). The liver samples of mice were washed with PBS twice and then cut into small pieces of 3-4 mm3. Five pieces were immediately put in a disposable disaggregator Medicon with 50 μm separator mesh (Becton Dickinson, USA) and then 1 ml PBS was added and processed in the Medimachine System for 1 min. Disaggregated cells were removed and pressed through 70 μm cell strainers to obtain single cell suspensions. Cells were used immediately for flow cytometric analysis. Related antibodies were listed as follows: CD45 (557235, BD Pharmingen, USA), CD11b (557397, BD Pharmingen, USA), Gr1/Ly6C (560595, BD Pharmingen, USA), Ly6G (551460, BD Pharmingen, USA), and F4/80 (25-4801, eBioscience, USA). Flow cytometric analysis was performed on a FACS Aria II (BD Bioscience, USA).
3
Isolation of Neutrophil Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh tumors and biopsies were mechanically dissociated with a BD Medicon (BD Bioscience), filtered, and washed once with PBS 1X.
Low density neutrophils (LDN) and high density neutrophils (HDN) were isolated from whole blood through Histopaque-based density gradient centrifugation. Whole blood was layered on top of a solution of equal volumes of Histopaque-1077 and Histopaque-1119 (Sigma-Aldrich), in a 1:1 (blood: Histopaque) ratio and centrifuged at 2000 rpm for 20 min, without brake. Circulating LDN, possibly present in the peripheral blood mononuclear cells (PBMCs) layer of the peripheral blood and HDN, present in the granulocytes fraction, were collected for immunophenotyping by flow cytometry and functional assays. The plasma was collected for ELISA assays.
Low density neutrophils (LDN) and high density neutrophils (HDN) were isolated from whole blood through Histopaque-based density gradient centrifugation. Whole blood was layered on top of a solution of equal volumes of Histopaque-1077 and Histopaque-1119 (Sigma-Aldrich), in a 1:1 (blood: Histopaque) ratio and centrifuged at 2000 rpm for 20 min, without brake. Circulating LDN, possibly present in the peripheral blood mononuclear cells (PBMCs) layer of the peripheral blood and HDN, present in the granulocytes fraction, were collected for immunophenotyping by flow cytometry and functional assays. The plasma was collected for ELISA assays.
4
Neutrophil Isolation from Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh tumors and biopsies were mechanically dissociated with a BD Medicon (BD Bioscience), filtered and washed once with PBS 1X.
Low density neutrophils (LDN) and high density neutrophils (HDN) were isolated from whole blood through Histopaque-based density gradient centrifugation. Whole blood was layered on top of a solution of equal volumes of Histopaque-1077 and Histopaque-1119 (Sigma-Aldrich), in a 1:1 (blood: Histopaque) ratio and centrifuged at 2000 rpm for 20 min, without brake. Circulating LDN, possibly present in the peripheral blood mononuclear cells (PBMCs) layer of the peripheral blood and HDN, present in the granulocytes fraction, were collected for immunophenotyping by flow cytometry and functional assays. The plasma was collected for ELISA assays.
Low density neutrophils (LDN) and high density neutrophils (HDN) were isolated from whole blood through Histopaque-based density gradient centrifugation. Whole blood was layered on top of a solution of equal volumes of Histopaque-1077 and Histopaque-1119 (Sigma-Aldrich), in a 1:1 (blood: Histopaque) ratio and centrifuged at 2000 rpm for 20 min, without brake. Circulating LDN, possibly present in the peripheral blood mononuclear cells (PBMCs) layer of the peripheral blood and HDN, present in the granulocytes fraction, were collected for immunophenotyping by flow cytometry and functional assays. The plasma was collected for ELISA assays.
5
Quantification of mCD44v6 and MTA1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples wetted with 1 ml 1X PBS were mechanically disaggregated by placing them into a Medicon (BD Biosciences) and inserted in the Medimachine (BD Biosciences) for 45 sec at 100 rpm. Cell suspensions were filtered using a Filcon (BD Biosciences), washed twice with 1X PBS and divided at a concentration of approximately 1×107 cells/ml into aliquots for staining. For detection of mCD44v6 positive cells, cell aliquots were incubated with CD44v6 (Bio-Rad MCA1730, dilution 1:500) for 1 h at room temperature (RT) in an orbital shaker at low speed. Cells were then washed and the percentage of cells expressing mCD44v6 was detected following a 60 min incubation with a goat anti-mouse IgG1: FITC secondary antibody (Bio-Rad STAR132F; dilution 1:100).
For intracellular labelling, cells fixation-permeabilization was performed using 1% paraformaldeyde-1% saponin in 1X PBS for 20 min at 4°C before cells were incubated with MTA1 antibody (SantaCruz Biotechnology SC-17773 dilution 1:500) and stained with a Polyclonal Goat anti-mouse-RPE Goat F(ab)2 secondary antibody (Dako R0480, dilution 1:100). Unstained cells were processed in parallel and included as references during analysis as well as cells stained only with the secondary antibodies. Flow cytometry analysis was performed using a Coulter Epics XL-MCL Flow Cytometer and its integrated SYSTEM II™ Software.
For intracellular labelling, cells fixation-permeabilization was performed using 1% paraformaldeyde-1% saponin in 1X PBS for 20 min at 4°C before cells were incubated with MTA1 antibody (SantaCruz Biotechnology SC-17773 dilution 1:500) and stained with a Polyclonal Goat anti-mouse-RPE Goat F(ab)2 secondary antibody (Dako R0480, dilution 1:100). Unstained cells were processed in parallel and included as references during analysis as well as cells stained only with the secondary antibodies. Flow cytometry analysis was performed using a Coulter Epics XL-MCL Flow Cytometer and its integrated SYSTEM II™ Software.
6
Isolation and Characterization of Rectal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC were isolated from whole blood samples by standard density gradient centrifugation (Lymphoprep, Axis-Shield, Oslo, Norway), numbered and about 1 million of cells were stored at −80 °C until use. An immunophenotypical analysis was realized on the remaining fresh blood cells. Two out of four rectal biopsies were immediately frozen at −80 °C until molecular biology testing. The remaining fresh biopsies were dedicated to the immunophenotypical analysis and were first incubated in RPMI 1640 (PAA, Pasching, Austria), supplemented with 1 % of antibiotics (penicillin 100 U/ml and streptomycin 100 µg/ml, PAA) and 10 % of fetal bovine serum (Sigma Aldrich, Saint-Louis, USA). Biopsies were then crushed immediately with a sterile scalpel and a medicon of 50 µm (BD Biosciences, San Jose, USA) and 1 ml of EDTA/Versene solution (Gibco Life technologies, Paisley, UK) was added to the cellular suspension to avoid aggregates. A mechanical disruption and a filtration of the suspension were performed with a syringe equipped with a 30-gauge blunt-end needle (Nipro Europe, Zaventem, Belgium) and a sterile filter of 30 µm (BD Biosciences). Then, rectal cells were washed with RPMI-FCS and re-suspended in that medium. The quantity of cells was determined by the automated cell counter TC10 (Biorad, Hercules, CA, USA), and the viability was estimated by a trypan blue labelling.
7
Evaluating TLR2 and TLR4 Expressions in CL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Skin samples were obtained from CL patient lesions using a 4-mm punch. Biopsies were treated with collagenase for 90 mins (37°C, 5% CO2), dissociated and cell suspensions were filtered using a 50 μm Medicon filter (BD pharmingen). To evaluate TLR2 and TLR4 expression, surface molecules were labeled using anti-CD14 (APC) (eBioscience, San Diedo, CA, USA), anti-TLR2 PE (clone TL2.1) and anti-TLR4 PE (clone HTA125) (IMGENEX, San Diego, CA, USA). Receptor expression was analyzed by flow cytometry, with 200,000 events acquired per sample.
In addition, biopsies from L. braziliensis patients were cultured in complete RPMI medium in the presence or absence of 100uM anti-TLR2 and 100uM anti-TLR4 for 72 hours (37°C, 5% CO2). Biopsy culture supernatants were collected and stored at −70°C until the time of IL-10, IL-1β, TNF and CXCL9 quantification by ELISA (R&D Systems) in accordance with the manufacturer’s instructions. Expression results are expressed in pg/ml.
In addition, biopsies from L. braziliensis patients were cultured in complete RPMI medium in the presence or absence of 100uM anti-TLR2 and 100uM anti-TLR4 for 72 hours (37°C, 5% CO2). Biopsy culture supernatants were collected and stored at −70°C until the time of IL-10, IL-1β, TNF and CXCL9 quantification by ELISA (R&D Systems) in accordance with the manufacturer’s instructions. Expression results are expressed in pg/ml.
8
Isolation of Intestinal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ileum and colon biopsies were sampled. After a mechanic digestion, enzymatic digestion was performed with 10 U/mL of collagenase and 1/500 DNase (Sigma-Aldrich, St Quentin Fallavier, France) for 20 min at 37°C under agitation. Samples were ground in Medicon (BD Biosciences, Erembodegem, Belgium) and 30 μm filtered (BD Biosciences) after three washes. Cells were centrifuged at 200 × g for 5 min and resuspended into RPMI medium (RPMI 1640, 10% FBS, 1% penicilline/streptomycine, 2 mM L- glutamine, 1% non-essential amino acids, 10 mM Hepes, and 1 mM sodium pyruvate).
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!