Blood samples were collected from advanced breast cancer patients according to the IRB protocol approved by the University of Texas Health Science Center at San Antonio (CTRC# 07-32). About 15 mL of Ficoll (GE Healthcare Life Sciences) was placed in a 50 mL conical tube, and 8 mL of the blood sample was carefully added on the top of the Ficoll. CTCs remained in the interphase peripheral blood mononuclear cells (PBMCs) layer after centrifugation at 400× g at RT for 30 min. The PBMC layer was carefully moved into a 15 mL conical tube and mixed with 10 mL of the MojoSort buffer (Biolegend, San Diego, CA, USA). After centrifugation again at 200× g for 5 min at 4 °C, CTCs were cleaned by the MojoSort buffer. Then, CTCs were cultured and expanded in the PRIME-XV tumorsphere medium (Fujifilm Irvine Scientific, Santa Clara, CA, USA) for 1 week for enrichment. For collection, cells were dispersed and washed by the MojoSort buffer and contaminated blood cell depletion was carried out using 10 μL of Human CD45-nanobeads (Biolegend, San Diego, CA, USA). CD45− CTCs were harvested in the supernatant by a magnetic stand after a few washes and resuspensions with the MojoSort buffer.
Ficoll
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany, France, Canada, Japan
Ficoll is a laboratory reagent used for the separation and isolation of cells and other biological particles. It is a synthetic, high-molecular-weight, neutral, and hydrophilic polysaccharide that forms density gradients when centrifuged. This property allows for the separation of different cell types or other biological materials based on their density.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (32 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (28 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (26 mentions)
L glutamine, by Thermo Fisher Scientific (23 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (17 mentions)
Penicillin, by Thermo Fisher Scientific (14 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (12 mentions)
Streptomycin, by Thermo Fisher Scientific (11 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Cd14 microbead, by Miltenyi Biotec (9 mentions)
Fetal calf serum (fcs), by Merck Group (8 mentions)
Percoll, by GE Healthcare (8 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (7 mentions)
Dextran t500, by GE Healthcare (7 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Merck Group (5 mentions)
Phosphate buffered saline (pbs), by Merck Group (5 mentions)
Hematology analyzer, by Beckman Coulter (5 mentions)
495 protocols using ficoll
1
Enrichment and Isolation of Circulating Tumor Cells
2
Isolation of Peritoneal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peritoneal lavage fluids were collected from patients operated at the Department of Visceral Surgery and Medicine, Inselspital Bern, Switzerland (Supplementary file 1A ). Peritoneal lavage fluid was filtered through a 100 µm filter and a Ficoll (GE Healthcare, #17-5442-02) gradient was performed by pipetting 10 ml Ficoll under 40 ml peritoneal lavage fluid in a 50 ml Falcon tube. Tubes were centrifuged (800 g, 4°C, 20 min, no brake). Cells were stained for flow cytometry as described below with anti-human antibodies (Supplementary file 1E ).
3
Yeast Vacuole Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast cells (i.e., the pep4Δ strain expressing Vac8-3xmCherry) were grown to an OD600 of ∼0.9 to 1.0 in 1 liter of YPD medium. After harvesting cells by centrifugation (5 min, 2,000 g), pellets were resuspended in buffer containing 0.1 M Tris/HCl, pH 9.4, and 10 mM DTT, and incubated for 10 min at 30°C. Cells were centrifuged as before and resuspended in the 25-ml spheroplasting buffer containing 0.3 mg/ml lyticase and then incubated for 25 min at 30°C. Spheroplasts were collected by centrifugation at 1,000 g for 3 min at 4°C and resuspended in 2.5 ml 15% Ficoll (15% wt/vol Ficoll in 0.2 M sorbitol and 10 mM Pipes/KOH, pH 6.8 [GE Healthcare]). After slow resuspension, cells were incubated with 0.02 mg/ml DEAE Dextran (Sigma-Aldrich) on ice for 5 min and then heat shocked for 2 min at 30°C. These suspensions were transferred to SW40 tube (Seton), and sequential layers of Ficoll solution in the aforementioned buffer (0.2 M sorbitol and 10 mM Pipes/KOH, pH 6.8; 3 ml of 8%, 3 ml of 4%, fill with 0%) were slowly added on top of the suspensions. Gradients were centrifuged at 100,000 g for 90 min at 4°C in an SW40 rotor. Vacuoles were collected at the 0–4% interface. The concentration of vacuoles was measured in the same way as described for purified autophagosomes.
4
Neutrophil Isolation from Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were isolated from peripheral blood using Ficoll–Paque gradient centrifugation and dextran sedimentation (Ficoll, GE Healthcare, Munich, Germany; dextran, Alfa Aesar, Tewksbury, MA, EEUU), as described previously (Salamone et al. 2010 (link)). Cells were resuspended in RPMI-1640 supplemented with either 1% FBS (only for apoptosis assays) or 10%; contained > 98% neutrophils (since most PMN are neutrophils, the terms will be used interchangeably from now on). Only samples with less than 0.50% monocytes were used (Supplementary Fig. 2).
5
Leishmania donovani Infection of Bone Marrow-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Promastigotes of L. donovani (MHOM/ET/67/Hu3:LV9) freshly differentiated from splenic amastigotes were cultured in Leishmania medium (M199 medium supplemented with 10% heat-inactivated FBS (Hyclone), 100 μM hypoxanthine, 10 mM Hepes, 5 μM hemin, 3 μM biopterin, 1 μM biotin, penicillin (100 I.U./ml), and streptomycin (100 μg/ml)) at 26 °C. For BMDM infections, metacyclic promastigotes were isolated at 1400 RPM in 15-ml Falcon conical centrifuge tubes containing 1 ml of 40% Ficoll (GE Healthcare) at the bottom, overlaid by 2 ml of a single gradient of 10% Ficoll and overlay 1 × 108 promastigotes from the late stationary growth phase in 2 ml of nonsupplemented DMEM (90 (link)). Complement opsonization of metacyclic promastigotes was performed prior to infections by incubating the parasites in HBSS containing 10% serum from DBA/2 mice for 30 min at 37 °C. BMDMs were then incubated at 37 °C with metacyclic promastigotes (parasite-to-macrophage ratio of 5:1). After 3 h of incubation, noninternalized parasites were washed 3× with warm HBSS. The time points are described in each experiment. Infection levels were assessed by microscopic examination of infected cells after Giemsa staining with the Hema 3 system (Fisher Scientific).
6
Induction of PBL Apoptosis by CPT
7
Peripheral Blood Mononuclear Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
8
Isolation and Storage of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Forty ml of blood were obtained weekly from the jugular vein of each animal (control and infected groups), starting from one day prior to the experimental infection. PBMCs were isolated before infection and 2 and 8 weeks post infection (WPI), as described previously [22 (link)]. Briefly, blood was collected in the presence of 100 μl of heparin (5000 units/ml) and centrifuged for 20 min at 600g, the buffy coat was collected and then diluted with 10 ml of phosphate-buffered saline (PBS) before being under-laid with 2 ml of Ficoll (1.077 specific gravity; Pharmacia Biotech, Sweden) and centrifuged for 30 min at 100g. The lymphocytes were collected and washed four times in PBS and stored in RNAlater® (Life Technologies, United States) following the manufacturer’s instructions.
9
ETOP-induced DNA Double-Strand Breaks Assay
10
Isolation and Culture of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 5-mL bone marrow sample was derived from the ileum of each animal and suspended in 5 mL phosphate-buffered saline (PBS) inside a sterile 15-mL conical tube. The sample was centrifuged at 1500 rpm for 20 min, then the buffy coat was isolated in 5 mL Ficoll (GE Healthcare, USA). The purified cells were harvested and washed twice using aseptic PBS. The samples were centrifuged again, then the supernatant was removed. The cells were resuspended (at the appropriate cell density) in 6-well plates. MSCs were cultured in a mixture of Dulbecco’s modified Eagle’s medium, 100 U/mL penicillin (Gibco, USA), 100 μg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco) in a humidified incubator at 37°C and 5% CO2. The culture medium was changed every two days. All the experiments were performed using MSCs harvested from passages 3 and 5.
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!