The permission from the dairy farm to use the blood samples from their animals for study purpose was obtained. Blood samples from pigs were obtained either at a local slaughterhouse, or from the chair for molecular animal breeding and biotechnology of Badersfeld. Venous whole blood was collected in sodium-heparin (25.000 I.U.) coated tubes and PBMC were prepared as previously described [5, (link)28] . Briefly, cow blood was diluted 1:2 in PBS (NaCl 136.9 mM, Na 2 HPO 4 × 2H 2 O 8.1 mM, KH 2 PO 4 1.4 mM and KCl 2.6 mM; pH 7.4) and PBMC were isolated by density gradient centrifugation (23 • C, 500× g, 25 min, brake off) using Pancoll separating solution (PanBiotech, Aidenbach, Germany). PBMC were obtained from intermediate phase, washed twice in PBS, and immediately used for in vitro stimulation. Pig PBMC were isolated by density gradient centrifugation (23 • C, 500× g, 25 min, brake off) of undiluted venous whole blood using Pancoll separating solution (PanBiotech, Aidenbach, Germany). PBMC were obtained from intermediate phase, washed twice in PBS (NaCl 136.9 mM, Na 2 HPO 4 × 2H 2 O 8.1 mM, KH 2 PO 4 1.4 mM and KCl 2.6 mM; pH 7.4), and immediately used for in vitro stimulation.
Pancoll separating solution
Manufactured by PAN Biotech
Sourced in Germany
Pancoll separating solution is a sterile, endotoxin-tested, density gradient medium used for the separation and isolation of mononuclear cells from whole blood or buffy coat. It is a non-toxic, isosmotic solution composed of polysucrose and sodium diatrizoate.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Lonza (3 mentions)
Easysep direct human neutrophil isolation kit, by STEMCELL (2 mentions)
Penicillin streptomycin, by PAN Biotech (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Gentamicin, by PAN Biotech (1 mentions)
L glutamine, by PAN Biotech (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Minimum essential medium (mem), by Lonza (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Na2hpo4 2h2o, by AppliChem (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Interleukin 7 (il 7), by Miltenyi Biotec (1 mentions)
Il 15, by Miltenyi Biotec (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Foetal calf serum, by Harvard Bioscience (1 mentions)
Rpmi medium, by PAN Biotech (1 mentions)
11 protocols using pancoll separating solution
1
Isolation of Bovine and Porcine PBMCs
2
Isolation of PBMCs from Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples (20 mL) in K3 EDTA tubes from 6 healthy volunteer donors from the national blood bank of Lomé were collected, and peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll density gradient centrifugation method described by Katawa et al.21 (link) In brief, 20 mL of whole blood was diluted in 15 mL of DPBS and carefully added to 15 mL of Pancoll separating solution (PAN-Biotech). After 20 minutes’ centrifugation at 2,000 rpm, the white layer of PBMCs was collected and washed 3 times in Roswell Park Memorial Institute (RPMI) medium supplemented with gentamicin 50 μg/mL, penicillin-streptomycin 100 μg/mL, L-glutamine 2 mM/mL, and fetal bovine serum (FBS) at 10% (PAN-Biotech). Cells were counted, and their viability was assessed using the trypan blue exclusion method.
3
Lymphocyte DNA Damage Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were taken 30 min after irradiation in the postoperative irradiation groups for staining of DNA double-strand breaks in lymphocytes as previously described [19 (link)]. Briefly, lymphocytes were isolated using Pancoll Separating Solution (Pan Biotech, Aidenbach, Germany). After separation, the lymphocytes were incubated overnight with the antibody detecting the phosphorylated variant of the histone H2AX (γ-H2AX) (Purified anti-H2A.X Phospho (Ser139); BioLegend, San Diego, CA, USA).
4
Neutrophil Isolation from Peripheral Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils and erythrocytes were separated from PBMCs by density gradient centrifugation (400g, 30 min, 15 °C, without brake) on Pancoll separating solution (density 1.077 g/mL; PAN Biotech, Aidenbach, Germany) from EDTA peripheral blood samples. To remove erythrocytes, the neutrophil pellet was mixed with one volume of phosphate-buffered saline (PBS; Lonza, Verviers, Belgium) and one volume of 6% dextran (Sigma-Aldrich, St. Louis, MO, USA) and erythrocytes were allowed to aggregate and settle during 30 min at 37 °C. Supernatant was collected and centrifuged (250g, 10 min, 15 °C). After washing the pellet with PBS, the remaining erythrocytes in the neutrophil pellet were lysed by hypotonic shock in distilled water for 30 s. After centrifugation and another two washing steps, the neutrophils were suspended in PBS and counted in a hemocytometer (> 95% neutrophils). Alternatively (patient II.5), granulocytes were isolated using the EasySep™ Direct Human Neutrophil Isolation Kit (StemCell Technologies, Grenoble, France). Both neutrophil isolation steps correlated perfectly in terms of neutrophil analysis afterwards (data not shown) and the choice for either method was based upon the number of samples to handle.
5
Isolation of Immune Cell Types
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD14+ monocytes were isolated from human 1-day-old buffy coats, derived from healthy donors (Blood Transfusion Center of the Red Cross, Mechelen, Belgium), via density gradient centrifugation on Pancoll separating solution (density 1.077 g/ml; PAN Biotech, Aidenbach, Germany) and via positive selection (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) (40 (link)). Neutrophils were isolated from fresh blood, derived from healthy donors, via density gradient centrifugation as described (21 (link)). Human embryonic diploid skin-muscle fibroblasts (E6SM cells) were grown in minimal essential medium (MEM; Lonza, Verviers, Belgium) containing 10% FCS.
6
Isolation of Peripheral Blood Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by gradient centrifugation. Blood was collected in EDTA S-Monovettes (Sarstedt). Collected blood was prediluted with RPMI 1640 (Thermo Fisher Scientific) and overlaid onto 15 ml Pancoll separating solution (PAN-Biotech). The tubes were centrifuged at a relative centrifugal force (rcf) of 1,600 for 15 min at room temperature with the deceleration of the centrifuge set to low. Isolated PBMCs were washed twice with RPMI 1640.
7
Neutrophil Isolation from Peripheral Blood
8
Isolation of Bovine Peripheral Blood Mononuclear Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bovine venous whole blood was collected in sodium–heparin (25.000 I.U., ratiopharm, Ulm, Germany)-coated tubes, and PBMC were prepared as previously described [5 (link)]. Briefly, blood was diluted 1:2 in PBS (NaCl 136.9 mM (Sigma-Aldrich, part of Merck-Millipore, Darmstadt, Germany), Na2HPO4 × 2H2O 8.1 mM (AppliChem, Darmstadt, Germany), KH2PO4 1.4 mM (Merck-Millipore, Darmstadt, Germany), and KCl 2.6 mM (Sigma-Aldrich); pH 7.2), and PBMC were isolated by density gradient centrifugation (room temperature, 290× g, 25 min, brake off) using Pancoll separating solution (PanBiotech, Aidenbach, Germany). PBMC were obtained from the intermediate phase, washed twice in PBS, and immediately used for in vitro stimulation.
9
Isolation of Rat PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were isolated as described before [21 (link)]. Briefly, rat full blood was diluted 1:3 with phosphate-buffered saline (PBS, Lonza Bioscience, Basel, Switzerland) and loaded onto Pancoll separating solution (density 1.077 g/mL, PAN-Biotech, Aidenbach, Germany). After centrifugation (1000 g, 10 min, no brake), the buffy coat was collected and the cells were washed twice with PBS.
10
Isolation and Transduction of Genetically Modified T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats of healthy donors (German Red Cross, Dresden, Germany) using density centrifugation with Pancoll separating solution (1,077g/ml) (PanBiotech, Aidenbach, Germany). Thereafter, primary T cells were isolated using pan T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The detailed transduction of the genetically modified RevCAR and Dual-RevCAR T cells were described in details previously (24 (link)). During transduction, T cells were cultured with IL-15, IL-7 and IL-2 (Miltenyi Biotec). However, 20-24 h before the assays, RevCAR T cells were cultured in RPMI complete medium lacking these cytokines. The research conducted with human T cells was approved by the local ethics committee of the Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Germany (EK138042014).
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!