Skip to Main contentScienceDirectJournals BooksRegisterSign in Sign inRegisterJournals BooksHelpLiberaseRelated terms:NeoplasmFollicleTransplantationEnzymePhosphate Buffered SalinePancreasDigestionNeutral ProteinaseCollagenaseView all TopicsDownload as PDFSet alertAbout this pageTumor Immunology and Immunotherapy – Integrated Methods Part BChun I. Yu, ... Karolina Palucka, in Methods in Enzymology, 20204.1 Processing single cell suspension from the spleen and lymph nodes1.In a Petri dish, a spleen is injected with 1 mL of enzyme solution containing 50 μg/mL of Liberase TM (Roche) and 24 U/mL of DNase I (Sigma) and incubated for 10 min at 37 °C. Similarly, lymph nodes are incubated with enzyme solution for 30 min.2.Prepare single cell suspension using two frosted slides to gently disrupt the spleen or lymph nodes and transfer the cell suspension into 15 mL conical tubes.3.Centrifuge 500 × g for 5 min and discard the supernatant.4.Lyse the RBC with 2 mL of RBC lysis buffer (Biolegend) for 5 min and wash once with 10 mL of 1 × PBS (see Note 23).5.Resuspend cells with 1 mL of 1 × PBS with 2% FCS and pass through a 70 μm cell strainer.6.Perform a cell count and proceed to downstream assays. Alternatively, samples can be cryopreserved in FCS with 10% DMSO in a controlled-rate freezing chamber, such as CoolCell (Corning) for 24 h in − 80 °C freezer and stored in the air-phase of a liquid nitrogen tank.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687919302423Tumor Immunology and Immunotherapy – Cellular Methods Part BAgnieszka Swierczak, Jeffrey W. Pollard, in Methods in Enzymology, 20204.2 Materials4.2.1.DMEM4.2.2.60 mm petri dishes4.2.3.Razor blades/scalpel blades4.2.4.15 mL conical tubes4.2.5.50 mL conical tubes4.2.6.PBS4.2.7.DNAse (Sigma-Aldrich)4.2.8.Liberase TL and Liberase DL (Roche)4.2.9.100 μm strainers/filters4.2.10.Human AB serum (Sigma-Aldrich)4.2.11.DAPI (Biolegend)4.2.12.5 mL Polypropylene FACS tubes4.2.13.5 mL 35 μm filter top FACS tubes4.2.14.Trypan blue4.2.15.Sorting antibodies (see Table 5)4.2.15.1.These are the minimum Markers we recommend for sorting neutrophils. The panel set up will depend on the machine specifications available hence we are not providing any fulorochrome recommendations. All panel design must first be checked against the specifications of the machines available and verified as workable by flow facility staff.Table 5. Antibodies required for isolation of tumor/tissue neutrophils.AntibodyCloneCell surface markerCD3UCHT1T cellsCD11bICRF44Myeloid cellsCD45HI30LeukocytesCD294BM16EosinophilsCD15aW6D3NeutrophilsCD163GHI/61TAMsaIt is recommended that a class IgG1 CD15 antibody is used as other classes can result in non-specific binding.4.2.16.Phenotyping antibodies (see Table 6)4.2.16.1.These panels were set up for use on a BD 6-laser Fortessa flow cytometer and are only meant to be used as a guide. All panel design must first be checked against the specifications of the machines available and verified as workable by flow facility staff.Table 6. Antibodies required for phenotyping tumor/tissue neutrophils.GENERAL PANEL: Used for gating on neutrophilsMarkerCloneFluorochromeCell typeConcentrationCD45HI30AF700Leukocytes1:200CD11bICRF44APC-Cy7Myeloid cells1:200HLA-DRL243FITCMonocytes1:100CD294 (Dump)BM16PECF594Eosinophils1:200CD3 (Dump)UCHT1PECF594T cells1:200CD19 (Dump)HIB19PECF594B cells1:200CD56 (Dump)B159PECF594NK cells1:100CD15aW6D3BV510Neutrophils1:100PANEL 1: Neutrophil phenotypic markersCD32FUN-2PE↑ Upon degranulation1:50CD63H5C6BV421↑ Upon degranulation1:50CD88S5/1PerCP-Cy5.5↑ Upon activation1:50CD89A59APC↑ Upon activation1:100CD66bG10F5PE-Cy7↑ Upon activation1:50PANEL 2: Neutrophil phenotypic markersCD54HA58APC↑ Upon activation1:50CD200ROX-108PEImmunomodulatory1:50CD274MIH2APCT cell co-stimulation1:50CD43CD43-10G7PE-Cy7↓ Upon activation1:50CD62LDREG56BV421↓ Upon activation1:50aIt is recommended that a class IgG1 CD15 antibody is used as other classes can result in non-specific binding.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687919303039Surgical techniques for total pancreatectomy and islet autotransplantationKaylene Barrera, ... Rainer W.G. Gruessner, in Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, 2020Islet isolationIf an in-hospital islet laboratory is available, the pancreas is processed and packaged on the back table.Alternatively, if an outside facility is used, the pancreas is submerged in UW preservation solution and chilled at 4 °C. Any preexisting ductal stents are removed and sent for culture. The pancreas is then packaged per UNOS guidelines in three bags.A total yield of  300,000 IEQ or  3000 IEQ/kg is desirable, however, patients have been shown to achieve insulin dependence even with lower yields.The isolation procedure as described by Ricordi remains to be the preferred method, with the use of Liberase which results in higher yields compared to the previously used collagenase P. A detailed description of the islet cell isolation is provided in this book in section \"Islet Isolation.” In brief, the pancreas is loaded into an isolator which consists of two stainless steel chambers with a mesh in between. Glass marbles are added to the chambers along with a solution of Liberase or collagenase. The chamber is connected to a shaker, and the digested pancreas passes through the mesh. This is then cooled to inactivate the enzymatic digestion. The solution is further filtered through a smaller mesh, and islets are purified using a series of centrifugation and gradients. The resultant pellets are then resuspended.4, 23View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780128148310000063Islet isolation for autotransplantation, following total or near total pancreatectomyElina Linetsky, Camillo Ricordi, in Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, 2020Collagenase: Selection and doseTranslating islet isolation technique that worked well in small animal models to large animal models and human organs proved to be difficult. A major obstacle to successful human and canine pancreas dissociation was the low enzymatic activity of the bacterial collagenase. Additionally, a number of reports indicated that a combination of collagenase and protease enzymes is necessary for best tissue dissociation and successful recovery of islets from large animal and human organs.20,59–61 The development of Liberase HI (Roche, Indianapolis, IN) in 1994, a highly purified enzyme blend that consisted of two collagenase isoforms from C. histolyticum (class 1 and class 2 collagenases) and a NP with very low endotoxin activity, has helped eliminate lot-to-lot variability in enzymatic activity. The use of Liberase HI resulted in consistently superior islet yield and quality compared to that obtained when unpurified collagenase products were utilized.20,59,60 Liberase HI also provided a convenient formulation in terms of its packaging, based on which a single vial was dissolved in a desired enzyme solution volume. The formulation of Liberase HI was later changed in such a way that two highly purified collagenase isoforms, class 1 and class 2 collagenases were packaged separately from NP. Using this enzyme combination, most centers utilized collagenase: NP in 1:1 ratio.21,62FDA’s request for better characterization of tissue dissociation enzymes utilized during the manufacture of human allogeneic islet cells in clinical trials resulted in withdrawal of Liberase HI from the market. A difficult transition from Roche Liberase HI to a different cGMP-grade enzyme combination, NB-1 collagenase (NB-1) supplemented with NB NP manufactured by SERVA Electrophoresis GMBH (Heidelberg, Germany) ensued. The latter was utilized during the National Institutes of Health (NIH)-funded Clinical Islet Transplantation (CIT) consortium trials, starting in 2007. Several transplant centers found that Serva’s NB-1 collagenase (NB-1) and NB NP (NB) mix failed to provide islet yields comparable to those obtained using Roche Liberase.62–65 This led to modifications to islet isolation protocols to increase islet yields, including selecting specific lots of Serva enzymes or modifying the sequence of enzyme addition used during the islet isolation procedure.62,63,65At the present time, several purified recombinant collagenase preparations are available from Roche (Indianapolis, IN), Serva (Heidelberg, Germany), and VitaCyte (Indianapolis, IN). These are utilized with the original NP or in combination with NP manufactured by a different company.21,66 Szot et al. demonstrated that using a mixture of cGMP-grade collagenase NB-1 and NB NP (SERVA Electrophoresis GMBH, Heidelberg, Germany) and a modified islet isolation process to account for the activity of Serva collagenase, consistently resulted in high-quality islet cell preparations suitable for clinical transplantation.63 Balamurugan reported superior islet cell yield, and improved islet cell structural integrity and quality, using VitaCyte collagenase with Serva NP.66 This enzyme combination was reported to produce total islet yields of  200,000 IEQ in 90% of attempted autologous isolations.66A dose of collagenase utilized for digestion varies between centers. While some centers utilize collagenase at a constant concentration63,66,67 disregarding the weight of the organ, others tailor collagenase dose according to the pancreas morphology and weight, which was shown to result in more consistent islet cell yields. For example, UMN varies the collagenase dose from 22 to 30 Wunch units/g and NP dose from 1.5 to 3.0 DMC units/g of pancreas weight, depending on the organ characteristics and weight.68For islet isolation from resected pancreata from CP patients, we have utilized Liberase MTF (recombinant collagenase, Indianapolis, IN) and Serva Collagenase-AF-1 (recombinant collagenase, SERVA Electrophoresis GMBH, Heidelberg, Germany) collagenase, both supplied as cGMP grade. We have found that a combination of either Liberase MTF (Indianapolis, IN) and Serva-AF-1 (SERVA Electrophoresis GMBH, Heidelberg, Germany) with NP-AF (SERVA Electrophoresis GMBH, Heidelberg, Germany) results in superior islet cell quantity and quality. We normally utilize 20–22 U/g of collagenase with 1.8–2.0 U/g of NP-AF, increasing the dose of NP-AF for severely fibrotic and damaged organs. For larger organs, we recommend that the volume of the enzymatic solution and the amount of both collagenase and NP be adjusted accordingly. We normally prepare the enzyme in 400 mL HBSS with 10 U/mL heparin, although the volume of the enzyme solution can be adjusted up for larger organs.66View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B978012814831000004XExtraction Techniques and Applications: Biological/Medical and Environmental/ForensicsS. Rafati, ... G.R. Rayat, in Comprehensive Sampling and Sample Preparation, 20123.03.2.3 Isolation of Adult Porcine IsletsThe common aim of all the islet isolation protocols is to obtain a high yield of viable islets with normal structure and function. Enzymatic digestion using proteolytic enzymes targets specific collagen type in the intracellular matrices, adhesive surface molecules, and cellular tight or gap junctions in the pancreas. Collagenase type XI and purified Liberase are forms of collagenase that are used in different concentrations in several types of solvents and in various temperature settings involved in protocols for islet isolation. Protective agents, such as bovine serum albumin (BSA), are used to coat the cells and prevent adhesion to each other and to the vessel wall. Minimum essential medium (MEM, developed by Harry Eagle) at 4 °C containing 20% newborn calf serum (NBCS) or fetal bovine serum (FBS) and also NBCS alone are usually used after incubation to inhibit further action of collagenase, preventing fragmentation and over digestion. Islet transplantation involves a complex sequence of pancreas procurement, organ digestion, and purification of released islets, quality assessment, culture, and final transplantation. In this method, dispersed islets throughout the gland are released by a mechanically enhanced enzymatic digestion process from the surrounding connective and exocrine tissue without destroying cluster integrity, and are then purified through density gradients designed to discriminate islets from contaminating nonendocrine cells. The isolation of adult porcine islets is similar to that of humans and porcine-specific difficulties of islet isolation are attributable to the intrinsic fragility of adult islets during the digestion of the pancreas.Under sterile conditions, the adult porcine pancreas is removed after exsanguination, and preserved in 4 °C Euro-Collins solution. Cold storage is used to preserve the islet integrity during efficient pancreas dissociation. In the operating room, the pancreas is cleaned from fat, lymph nodes, and vessels then dissected in two portions (head and body-tail) by cutting it at the neck portion. The pancreatic duct is cannulated and collagenase at 30 °C HBSS is delivered by syringe or controlled perfusion, which improves collagenase delivery throughout the pancreas.31,32 The pre-warmed (30 °C) HBSS containing 0.75 mg ml−1 Liberase PI is used to obtain retrograde distension of body and tail and ante-grade distension of the head. An appropriate distension of the organ is critical for successful islet isolation. In the islet processing laboratory, the distended pancreas is placed in a re-circulating digestion chamber, Ricordi chamber, or modified Ricordi chamber (with two stainless steel compartments separated by a mesh and seven one-cm glass marbles inside)27 which is set to a gentle periodical shaking, since adult porcine islets are more fragile than human islets. The circuit is filled with warm (30 °C) enzyme solution that circulates at a flow rate of 100 ml min−1 through a peristaltic pump. The chamber is then placed on the arm of the shaking device set to five oscillations every 2 min (10-cm excursion). The time of digestion varies, but, in general, it is carried on for about 16–25 min, maintaining the temperature at approximately 30 °C. The samples are collected during the digestion of the pancreas to verify the progression of the digestion process. When most islets are free from the surrounding acinar tissue and still intact, the digestion is stopped by diluting the enzyme solution in excess volumes of cold (4 °C) Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS at a flow rate of 320 ml min−1. The dispersed pancreatic tissue is collected and then washed with RPMI medium supplemented with 10% FBS before proceeding to the purification steps.The purification procedure of adult porcine islets is performed at 4 °C. Large-scale islet purification can be obtained using discontinuous or continuous gradient with the semi automated COBE 2991 cell processor. COBE’s name was derived from the first two letters of the founders’ last names of the COBE Company, producer of blood component machines. COBE was initially designed for blood cell separation in blood clinics and later used for islet purification. Thiery B. et al.35 described the protocols for the isolation of adult porcine islets using both discontinuous and continuous gradients. Briefly, in the discontinuous density gradient, the pancreatic digest is suspended in Euro-Collins–Ficoll gradient with a density of 1.121 g cm−3 and the digest is then transferred into a bag. A standard sterile cell processing set is used with a peristaltic pump connected to the red COBE line, which will be used to load the gradients. The transferred bag is attached to the green line, and the digest is loaded by gravity into the processing bag. The centrifuge is spun at 2000 rpm, and the air is vented from the processing bag by releasing the clamp from the green tube and opening the supernatant out valve. Then, the outputs are clamped and the COBE 2991 is stopped. At this point, the centrifuge is then spun at 1200 rpm, and Euro-Collins–Ficoll gradients with various densities are loaded from the red line. After a 3–5-min spin, the interfaces are collected through the red line. Three fractions are collected where the first layer is discarded (100–125 ml): fraction 1, which contains the highly pure islets, fraction 2, which contains fewer islets that are less pure, and fraction 3, contains the acinar tissue. The collected fractions are washed with RPMI, which contains 10% FBS, and samples are then taken to assess the number of islets and purity of the preparation prior to culture.The continuous gradients are performed using a gradient maker that is connected to the red line of the COBE 2991 processing bag. The gradient maker consists of two connected chambers, with the higher density chamber placed on a magnetic stirrer. The pancreatic digest is loaded either on top, which is suspended in the preservation solution or at the bottom, which is suspended in the high-density gradient of the processing bag. The continuous gradient is loaded by peristaltic pump and, after loading 120 ml of the high-density medium or the digest suspended in the high-density medium, the COBE 2991 bowl is spun and the air is released from the bag. The valve between the two chambers is then opened to obtain a linear dilution of the heaviest gradient with the lightest to form a continuous density gradient, which is pumped into the processing bag. When the whole gradient has been transferred, University of Wisconsin (UW) or HBSS with 2% FBS is loaded on top of it. The excess air is vented off, and the speed is increased (2000–2200 rpm) for 5 min to allow islet and acinar tissue to migrate to their respective densities. The gradients are then unloaded through the red line and collected into one 100–150-ml fraction (waste) and 9–11 smaller fractions (30 ml each). Each fraction is washed in RPMI with 10% FBS, and samples are taken for the determination of the amount and purity. Figure 4 shows a schematic diagram of the isolation method used for adult porcine islets.Figure 4. Schematic diagram of the method used for the isolation of islets from adult pigs.In 1999, Brandhorst H. et al. used only the splenic lobe of the pancreas due to the asymmetrical distribution of islet volume within the gland. Low-temperature digestion of porcine pancreas (24–28 °C) was then utilized with a ratio of 1.7 ml of UW solution per gram of pancreatic tissue containing 0.15% (w/v) Liberase HI (Roche–Boehringer–Mannheim, Indianapolis, IN), a highly active purified and defined enzyme blend. Significant progress included almost double the yield of purified islet equivalent per pancreas using Liberase HI compared to regular collagenase (526 480 ± 46 560, n = 48 versus 270 270 ± 19 420, n = 46; p ≤ 0.0001) and increased islet equivalents per gram of pancreas (4210 ± 320, n = 48 versus 2640 ± 245, n = 46; p = 0.0004).33 However, the purity, viability, and in vitro function of islets did not differ between the two groups. This experiment proved the superiority of Liberase HI in reproducibility of islet isolation outcome, determined by the higher yield and preservation of morphological and functional integrity of islets. In 1998, Cavanagh T.J. et al. did the first trial of the commercially available purified enzyme blend, Liberase PI (Roche Biochemical, Basel, Switzerland) for pig islet isolation.34 Liberase PI was developed specifically for porcine islet isolation, instead of regular crude collagenase to distend the pancreas intraductally, and reported significant progress in porcine islet isolation. Islet integrity was better preserved during digestion with Liberase PI compared with regular collagenase digestion. The refined enzyme solution in the form of Liberase PI allowed the digestion process of the pancreas to occur at lower temperature and concentration to prevent over digestion and fragmentation of nonliberated islets.In 2004, Kirchhof N. et al.14 utilized UW solution with 0.15% (w/v) Liberase PI to intraductally distend adult pig pancreas, which were then dissociated using the automated method at 28–32 °C. Liberated islets were separated from nonislet tissue using continuous UW/OptiPrep density gradients on a COBE 2991 cell separator. Isolated adult porcine islets were then cultured free-floating in Medium 199, supplemented with 10% donor adult porcine serum and ciprofloxacin for 48 h. The group reported an average islet yield of 341 207 ± 168 686 islet-equivalents, which corresponds to 1740 ± 325 islet-equivalents g−1 of pancreas. After 48 h of culture, the average islet yield was 69 ± 15% and the purity of the graft as assessed by the percentage of dithizone-positive cells was over 90% with a mean of 94%.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123813732000673Islet Transplants for Diabetes: The Edmonton ProtocolPeter A. Senior BMedSci, MBBS, Ph.D., MRCP (UK), A.M. James Shapiro MBBS, Ph.D., FRCS, FRCPC, in Cellular Transplantation, 2007ISLET ISOLATION AND CULTUREEven though the pancreas is a sensitive organ, the process of islet isolation [102] requires the pancreas to be subjected to a hostile environment. The initial stage involves the cannulation of the pancreatic duct and controlled distension of the pancreas with collagenase [72]. Subsequently, the pancreas is cut into small pieces and placed in the Ricordi chamber, a stainless steel container, along with stainless steel marbles. The chamber is perfused with a blend of enzymes at 37°C and agitated. With this combination of chemical and mechanical dissociation, islets are freed from exocrine tissue. Digestion of the pancreas is generally complete within less than 30 minutes. Islets are separated from exocrine tissue over a series of density gradients using a COBE cell separator. Generally, the final islet preparation consists of 5 cc of tissue.The use of liberase as the digestive enzyme for islet isolation represented an advance over the commercially available collagenases used previously, which exhibited substantial variability in enzyme activity between batches [75]. Liberase is a purified blend of collagenase isoforms 1 and 11. Earlier reports suggested that there was good reproducibility between Liberase lots [70]. Nevertheless, substantial variation in islet yields persisted. More recently, evaluation of collagenase blended with a neutral protease at the time of use has yielded promising results [23].Prior to release, islets are subject to a number of quality control tests, including a gram stain to rule out bacterial contamination and an assay to exclude significant amounts of endotoxin. Further assays of biological efficacy can also be employed, including measurement of insulin content, insulin secretion in response to low and high glucose (stimulation index), or bioassays such as transplantation into diabetic mice. The release criteria are outlined in Table 5.1.TABLE 5.1. Release criteria for clinical islet transplantation.ParameterCut PointIslet number 4,000 ie/kgPacked cell volume 10 ccIslet viability 70%Endotoxin content 5 EU/kgGram stainNo bacteria seenPreviously, freshly isolated islets were suspended in transplant medium and infused immediately [121]. Long-term culture of islets is associated with a loss of endocrine cells [115]. However, it is clear that islets can be successfully cultured for several days prior to transplantation without significant loss of potency [56]. Indeed, the ability to culture islets prior to transplantation has a number of advantages. Safety is improved because transplants can be scheduled when the entire transplant team can be present. Time is also available to administer conditioning or other immunosuppressive therapy, avoiding exposure of islets to the cytokine release associated with many cell-depleting induction therapies. Logistically, patients no longer need to live close to the transplant center for indeterminate periods of time. Indeed, islets can be successfully shipped from the isolation facility to distant transplant centers [48]. This opens up the potential for a small number of core islet isolation facilities, which would likely have economies of scale because islet isolation must be performed in a cGMP facility. Preliminary data from a multicenter trial of islet transplantation indicate superior outcomes from experienced centers and confirm the steep learning curve associated with islet isolation [120, 123]. Thus, the concentration of expertise in a small number of facilities seems sensible.Although there is generally some loss of islets during the culture period, there is an increase in islet purity because of more rapid loss of exocrine tissue. This increased purity, along with the associated reduction in the volume of tissue to be infused, has further safety advantages. Smaller and more pure preparations are associated with a reduced risk of portal hypertension and portal thrombosis. Thus, the ability to culture islets prior to transplant has a number of benefits both in terms of practicality and safety.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123694157500065Assessment of Changes in the Peripartum CervixYucel Akgul, Mala Mahendroo, in The Guide to Investigation of Mouse Pregnancy, 2014Reagents•Staining buffer (SB): 2% fetal bovine serum and 7.5 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) in PBS•Digesting buffer (DB): for 5 ml of SB add:•200 μl of Liberase TM Research Grade (equal to 28 Wunsch units/ml, Roche)•12.5 μl of 1 mg/ml DNase I (Sigma–Aldrich)•10 turbidity-reducing units of hyaluronidase (Seikagaku)•1% paraformaldehyde (Sigma cat# P-6148) in SB•BD FACS™ lysing solution (BD Falcon, cat# 349202) diluted from 1 to 10 in water•Fluorochrome-conjugated primary antibodies such as Neutrophil (Neu) 7/4-PE (Serotec); F4/80-allophycocyanin, Cluster of Differentiation (CD) 45-PE-Cy7, CD11b-Pacific Blue, CD4-PE, CD8-PECy5, CD3 Pacific Blue, and/or CD19-biotin (eBiosciences); and Gr-1 (Ly6C/Ly6G)-allophycocyanin-Cy7, Siglec F-PE,and Ly6G-FITC (BD Biosciences)•Fc-Blocking antibody (mAb 24G2; BD Biosciences)View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123944450000643The Zebrafish: Disease Models and Chemical ScreensMyron S. Ignatius, David M. Langenau, in Methods in Cell Biology, 2011V Confirming Transformation of Fluorescent-Labeled Tumor Cells by Cell Transplantation into Irradiated Recipient AnimalsOne of the most basic assays used in our laboratory is cell transplantation of fluorescent-labeled tumors into irradiated animals to assess if fluorescent cell populations are fully transformed (Fig. 3A). Sublethal-irradiation is commonly used to transiently ablate the immune system, providing engrafted cancer cells the ability to grow unabated by immune responses (Langenau et al., 2003; Traver et al., 2004). However, irradiation protocols provide only a transient assay for tumor growth, with animals capable of mounting robust immune responses after 21 days post-irradiation (Smith et al., 2010). To obviate these concerns, it is best to assess transplants prior to 20 days post-transplant and/or to transfer large numbers of tumor cells for engraftment where even after recovery of the immune system, the tumor cells overwhelm the immune system and cannot be cleared.Fig. 3. Cell transplantation assays in zebrafish. (A) Establishing malignancy by cell transplantation into irradiated recipients. A GFP+ primary tumor can be excised and individual cells isolated by disaggregation and filtering. Cells are induced into irradiated recipient animals (23 Gy, 2 days post-IR) and recipients assessed for engraftment from 7 to 21 days post-transplantation. (B) Identification of tumor-propagating cells. In this published example (Langenau et al., 2007), rag2-kRASG12D was co-injected with rag2-dsREDexpress into one-cell stage alpha-actin-GFP+ animals. Because the rag2 promoter expresses in satellite cells and early myoblasts while alpha-actin is expressed in late myoblasts and fused fibers, four cell populations could be identified following tumor resection, disaggregation, and fluorescent activated cell sorting (FACS). Each cell population is introduced into irradiated recipient animals and assessed for the ability to remake tumor. In this case, only the rag2-dsREDexpress+/alpha-actin-GFP-negative cells were capable of engraftment. (C) Self-renewal and single cell transplant assays using clonal, syngeneic zebrafish. In this example based on Smith et al. (2010), fluorescently labeled T-ALLs were created by microinjecting rag2-mouse cMyc (rag2-cMyc) and rag2-GFP into one-cell stage CG1-strain, syngeneic zebrafish. A subset of animals develop fluorescent-labeled T-ALLs that can be FACS sorted and transplanted at limiting dilution into CG1 recipient animals. Six animals per group were monitored for T-ALL onset up to 120 days post-transplantation. Limiting dilution cell transplantation assays accurately determine the number of leukemia-initiating cells contained within the bulk of the tumor mass and have been reviewed previously (Ignatius and Langenau, 2009). A single cell can also be transplanted into recipient fish, but fewer animals develop disease. (See color plate.)A MethodFluorescent-labeled zebrafish cancer models can be created as outlined above. Tumor-bearing animals are euthanized by tricaine overdose, and then the tumor is excised from the animal using a razor blade. For most applications, the tumor is dissected along with noneffected margins on one side to maximize the amount of tumor cells. On the opposite side of the animal, some of the tumor is left within the body and fixed in 4% paraformaldehyde overnight. This way, tumor histology can be completed on transplanted tumors.Most tumor cells are easily dispersed from resected tumor by repeatedly mincing the tissue with a razor blade in the presence of 250 μL of 0.9× PBS + 5% FBS. This volume can change based on the size of the resected tumor, but the volume should be limited to increase the efficiency of tumor cell extraction from the tissue. Usually, the tissue is minced 50 to 100 times for 30–60 s at room temperature in a 10 cm Petri dish. Following mincing, 0.9× PBS+ 5% FBS solution is added to the tumor samples to get a final volume of 10 mL. The sample is aspirated repeatedly 10 times using a 10 mL plastic pipette and a pipette aid. This procedure loosens tumor cells facilitating disassociating from normal tissue. Next, the sample is filtered over a 40-micron filter (BD Biosciences, 352340) into a 50 mL conical tube. The filter is then washed with 10 mL of 0.9× PBS+ 5% FBS. Some solid tumors can benefit from treating the samples with collagenase to release tumor from normal unaffected tissue. We have previously used liberase III from Roche to isolate ERMS cells from affected animals; however, this product has recently been replaced by liberase blendzyme III. We have not found an optimal dose for extracting tumor cells using the new liberase blendzyme III reagent. Therefore, we no longer use collagenase or liberase treatment to isolate embryonal rhabdomyosarcoma cells from tumor-bearing fish but still obtain sufficient numbers of tumor cells for most assays.A hemocytometer is used to count the numbers of cells contained in the tumor cell suspension by (1) vortexing the sample for 5 s at 75% velocity, (2) pipetting 10 mL of sample into 90 mL of trypan blue (Gibco, #15250-061), (3) vortexing this mixture for 5 s at 75% velocity, and (4) transferring 10 mL of the sample to a hemocytometer (Hausser Scientific, #1492). Cell counts are estimated by counting the number of optically clear cells within a 16 square viewing area and then multiplying the number of cells by the dilution factor (10) and then by 10,000 to arrive at the number of cells per mL. For some applications, unsorted cells can be transplanted directly and thus, cell counts can be used to directly make the suspension as concentrated as needed. If the sample is too concentrated, 0.9× PBS + 5%FBS can be added to the sample. If the sample is too dilute, the sample can be centrifuged at 1000G for 10 min, the supernatant removed, and the sample resuspended to the correct volume required. If pure populations of cells are required, FACS can be used to isolate homogenous fluorescent cells.Once tumor cell suspensions have been isolated, tumor samples can be introduced into irradiated recipient animals by intraperitoneal injection (5 mL of solution per fish using a #701N Hamilton Syringe, #80366). It is important to irradiate recipient animals 2 day prior to cell transplantation with 23 Gy from a Cesium-137 radiation source to transiently ablate the immune system (Traver et al., 2004). Some strains of fish are much more sensitive to gamma-irradiation, so dose optimization may be required to enhance engraftment of tumors and/or curb death due to irradiation. For example, we find that 23 Gy irradiation doses are lethal to optically clear caspar fish. For cell transplantation of unsorted tumor cells, we commonly utilize 1×106 T-ALL cells and 2×104 ERMS cells to engraft tumors into irradiated recipients (Langenau et al., 2003, 2007). Cell transplantation of lower numbers of tumor cells can often lead to robust engraftment (Smith et al., 2010).Imaging fluorescent tumor cell engraftment into irradiated recipients can be completed using an epifluorescence dissecting microscope or the LED fluorescence macroscope (see Section III). Transplant recipients are routinely monitored for engraftment at 7, 14, 21, and 28 days post-transplantation. Photographs can be taken at each time point to visualize fluorescent-labeled tumor growth within the affected animal over time. Engrafted animals are routinely sectioned to confirm tumor histology. Tumor cells can also be isolated from recipient animals and used in serial transplantation experiments to propagate the tumor and to verify long-term tumorigenicity of the target cell population. We caution that tumor growth after 21 days post-irradiation treatment will be greatly impacted by immune system recovery and tumor cells will often begin to be killed by the host. Although this assay is only a short-term assay for transformation, the power of cell transplantation into irradiated recipient fish lies in the ability of tumor-derived cells from any strain of fish to be efficiently engrafted into recipient animals, irrespective of immune matching.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123813206000199Tumor Immunology and Immunotherapy – Cellular Methods Part BAnnika M. Bruger, ... Pierre van der Bruggen, in Methods in Enzymology, 20203.3 MDSC preparation from solid tissueSamples from tumors or tumor biopsies are rare, difficult to obtain and usually small, which limits the expected MDSC yield. Moreover, solid tissue samples require the most manipulation for MDSC isolation, as the tissue has to be gently dissociated. MDSC are isolated from the mononuclear fraction following density gradient separation. A stain for CD45 is required, as epithelial or cancer cells could be present.a.We work with a hospital in very close proximity, and we receive tumor samples within 30 min after extraction. The specimens are placed in a 50 mL flask in PBS and transported on ice.b.Place the tumor and any accompanying PBS into a sterile Petri dish on ice underneath a flow hood. Cut the tumor into very small pieces (1–2 mm) using a sterile scalpel. The pieces should be small enough to not block a 10 mL pipette.c.Place the tumor pieces and fluid into a MACS dissociator C tube (Miltenyi, catalog ♯ 130-093-237). Add DNase I at 5 U/mL and Liberase™ TL and DL (thermolysin-dispase-low, both Sigma and Roche, catalog ♯ 05401020001 and LIBDL-RO) at 0.5 and 0.26 U/mL, respectively.d.Dissociate the tumor cells using the pre-set program \"h_tumor_01” on the gentleMACS™ dissociator (Miltenyi, catalog ♯ 130–093-235) for 45 s.e.Incubate the tumor cells in the dissociator tube under agitation for 45 min at 37 °C.f.Dissociate the tumor cells using the dissociator s pre-set program \"h_tumor_01” for 45 s.g.Transfer the tumor cells into an FBS-coated 50 mL Falcon tube. Rinse the dissociation tube with equal volume of human serum and add this to the tumor cells in the Falcon tube. The human serum stops the digestion by the liberase. Pellet the cells by centrifugation at 400g for 10 min.h.Resuspend the tumor cells with 50 mL PBS + 1 mM EDTA per Falcon tube and pass the cells successively through a 70 and 40 μm filters, washing the cells by centrifugation at 400g for 8 min in between. Count and collect the cells through centrifugation at 400g for 8 min.i.Resuspend the cells in warm RPMI 1640 to a total volume of 35 mL.j.Perform density gradient separation on the cell suspension as previously described in Section 2.1, steps b and c.k.Following the density gradient separation the mononuclear cells settle in a thin layer below the medium, and above the Lymphoprep solution and erythrocyte pellet (which is expected to be small in tumors). Carefully remove only the PBMC layer with a pipette and place it into an FBS-coated 50 mL Falcon tube.l.Wash the cells with warm PBS + 1 mM EDTA and centrifuge at 300g for 10 min.m.The PBMC settle in the pellet. Carefully remove the supernatant.n.Repeat the washing steps with 50 mL PBS + 1 mM EDTA twice.o.Refer to MDSC staining protocol in Section 3.4.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687919302277Flow Cytometric Analysis of Myometrial and Decidual Cell SuspensionsAdam Blaisdell, Adrian Erlebacher, in The Guide to Investigation of Mouse Pregnancy, 2014Equipment, Mice, and Supplies1.Equipmenta.Flow cytometer, LSRII (BD Biosciences), three-lasers (405, 488, 633 nm)b.Cell acquisition software (BD Biosciences FACSDiva)c.FACS data analysis software, such as FlowJo (Tree Star) or FCS Express (De Novo Software)d.Microcentrifuge, refrigeratede.Water bath, 37°C2.Dissection microscope and light source3.Instrumentsa.Dissection/mincing toolsScissors, straight, 14.5 cm (Fine Science Tools #14000-14)Scissors, extra narrow, 10.5 cm (Fine Science Tools #14088-10)Scissors, fine, curved, 10.5 cm (Fine Science Tools #14061-10)Graefe forceps, straight, 10 cm (Fine Science Tools #11050-10)Dumont #5 forceps, straight, 11 cm (Fine Science Tools #11251-20)b.Pipetters (1 ml, 100 μl, 10 μl) with tipsc.10 cm Petri dish (BD Falcon #351029)d.1.7 ml microcentrifuge tubes (Denville Scientific #C2170)e.Sterile disposable filter, polyethersulfone (PES) membrane (90 mm), 0.2 μm pore size (Thermo Scientific #569-0020)f.Cell strainer, nylon mesh, 70 μm (BD Falcon #352350)g.Hematocytometer and light microscopeh.5 ml FACS tube, polystyrene, round bottom (BD Falcon #352052)i.(Optional) Insulin syringe (Becton Dickinson #309301)4.Nonpregnant or pregnant mice, or mice with artificial deciduas5.Reagentsa.Hanks’ Balanced Salt Solution (HBSS) with Ca2+ and Mg2+ (Invitrogen #14025-076)b.Liberase TM Research Grade (Roche #05401127001), diluted to 28 Wunsch units (WU)/ml (100×) in double-distilled water (ddH2O) and stored in 50 or 100 μl aliquots at −20°C; thawed as needed and used immediatelyc.DNase I (Roche #10104159001), diluted to 30 mg/ml (1000×) in ddH2O and stored in 5–10 μl aliquots at −20°C; thawed as needed and used immediatelyd.(Optional) Trypsin, 2.5%, no phenol red (Invitrogen #15090-046, 50×) stored in 100 or 200 μl aliquots at −20°C; thawed as needed and used immediatelye.(Optional) Ammonium chloride potassium (ACK) lysing buffer (Lonza #10-548E)f.Phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Cellgro #21-031-CV)g.Bovine serum albumin (BSA; Sigma #A2153)h.Trypan Blue (Thermo Scientific #SV30084.01)i.FcγR blocking antibody (anti-CD16/32; Clone 2.4G2). This reagent is available from multiple vendors. We buy ours in bulk from BioXCell (#BE0008). The antibody is aliquoted at 1 mg/tube and stored at −80°C until needed, then diluted to 1 mg/ml in PBS and stored at 4°Cj.(Optional) RPMI 1640 medium (Invitrogen #21870-076)k.(Optional) Fetal bovine serum (FBS; Atlanta Biologicals #S12450), heat inactivated for 30 min at 55°Cl.(Optional) Heparin sodium, 1000 USP units/ml (Sagent Pharmaceuticals #400-01)6.Solutionsa.Tissue digestion buffer (example of 10 ml preparation)9.89 ml HBSS containing Ca2+ and Mg2+100 μl Liberase TM (28 WU/ml); final concentration 0.28 WU/ml10 μl DNase I (30 mg/ml); final concentration 30 μg/ml(Optional) 200 μl Trypsin (2.5%); final w:v 0.05%; subtract from HBSS volume accordinglyb.FACS buffer (example of 500 ml preparation)495 ml PBS without Ca2+ and Mg2+5 g BSA; final w:v 1%5 ml 0.5 M EDTA, pH 8.0; final concentration 5 mMFilter buffer through PES membrane with 0.2 μm pore size prior to use in order to sterilize and degas.c.(Optional) RPMI-10 (example of 500 ml preparation)450 ml RPMI 1640 medium50 ml FBSFilter buffer through PES membrane with 0.2 μm pore size prior to use.d.(Optional) Intravascular leukocyte labeling solution (100 μl per mouse)90 μl PBS without Ca2+ and Mg2+5 μl Heparin sodium, 5 USP units total5 μl anti-mouse CD45 (Clone 30-F11, 0.2 mg/ml), 1 μg total, preferably conjugated to a brighter fluorochrome such as PE (Biolegend #103105), PE-Cy7 (Biolegend #103113), or allophycocyanin (APC; Biolegend #103111)View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123944450000539Recommended publicationsInfo iconImmunityJournalJournal of Molecular and Cellular CardiologyJournalJournal of Allergy and Clinical ImmunologyJournalThe American Journal of PathologyJournalBrowse books and journalsAbout ScienceDirectRemote accessShopping cartAdvertiseContact and supportTerms and conditionsPrivacy policyWe use cookies to help provide and enhance our service and tailor content and ads. 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