Cell Culture Black

合集下载

SuperCulture

深圳市达科为生物工程有限公司地址：深圳市坪山区坑梓街道金辉路14号深圳市生物医药创新产业园区1号楼702、703518122SuperCulture ®MSC 专用基础培养基说明书产品描述SuperCulture ®MSC 专用基础培养基（Mesenchymal Stem Cell Basal Medium ，MSCBM ）是专用于培养人间充质干细胞（hMSCs ）的GMP 级别基础培养基，需要添加血清或者血清替代物混合成完全培养基后使用，推荐使用EliteCell 的EliteGro ™作为血清替代物添加。

产品信息产品名称货号型号规格Mesenchymal Stem Cell Basal Medium6114011A 型500mL 6114021B 型500mL*A 型表示有酚红，B 型表示无酚红。

产品参数产品名称Mesenchymal Stem Cell Basal MediumGlucose Low Glucose Supplements Need supplement Glutamine Glutamine Phenol Red Phenol Red AntibioticNo Antibiotic Sodium Bicarbonate BufferSodium BicarbonateHEPES BufferNo HEPES pH 7.0-7.6Osmolality 280~340mOsmol/kgEndotoxin<0.3EU/ml储存条件及有效期：MSC 专用基础培养基2℃~8℃避光储存，有效期为1年。

使用方法以配制500mL 完全培养基为例（注意以下均为无菌操作）：1.准备475mL 的MSCBM ，恢复到室温。

2.准备25mL 的EliteGro ™，37℃水浴解冻；3400xg 离心3~5min ，取上清部分，或者用40μm 的细胞筛网直接过滤。

3.将离心或过滤后的25mL EliteGro™加入475mL MSCBM，即可得到5%浓度的完全培养基（普通版本的EliteGro™还需要添加肝素钠，Advanced版本的EliteGro™不需要添加肝素钠）。

Mammalian Cell Culture 2011 (1)

• Form basis for multi-cellular life • More complex than prokaryotes
• Internal cellular structure compartmentalised • More fragile than prokaryotes
Setting up the culture
Culture must be established in sterile conditions!
• Any manipulation should be performed in a laminar flow hood • Awkward, but essential to ensure that cultures remain uncontaminated • All equipment must be sterilised before use:
• Established industry: many products passed through safety regulations • Primarily preventative and therapeutic products
• Cells are desired products (for cell delivery, tissue engineering in the next lecture)
Mammalian Cell culture
Dr. Liam Grover
Cell factories
• Cells can be used to produce a wide variety of chemicals and biopharmaceuticals • Microbes can be used to produce a variety of products and are comparatively easy to maintain

cell-culture(细胞培养前准备)

corridor
gate
cabinet Refrigerator
incubator
Sterile zone
Buffer zone
gate Liquor room
Gate
Super clean bench Bacterial free zone
Dressing zone
Buffer zone
Preparing room
Hanks:
salt solution
Super clean bench or Bio- safety Cabinet
CO2 cell incubator
Day1
Day2
Day3
Day4
Day5
Day6
Aseptic technique
Laboratory : 1. Cleaning the floor and surface of apparatus by benzalkonium bromide per week . 2. Sterilizing by ultraviolet before experiment . Human : 1. Cleaning hand and dress the aseptic clothes, hat ,mouth-muffle and slipper. 2. Sterilizing hand by 75% alcohol before operation .
Distilled water
Autoclave sterilizer
drying and baking Bench for package
Liquor room
Treatment of glassware

黑胶虫污染

• • •
Members of the Achromobacter and Alcaligenes genera have reclassified to and from these two genera, including Achromobacter (Alcaligenes) xylosoxidans and Achromobacter (Alcaligenes) denitrificans [22,23]. It is unclear whether the similarity to both members of the Achromobacter and Alcaligenes genera is due to uncertainty in the nomenclature of previously submitted sequences or due to the actual sequence of the contaminating bacteria. RDP, a web-based program containing only 16S rDNA sequences, was utilized to confirm the contaminating bacteria’s genus. Results using SeqMatch from the RDP identified the sequences as belonging to the genus Achromobacter. Phylogenetic trees based on the BLASTand RDP results grouped the bacteriawith predominantly Achromobacter 16S rDNA sequences (data not shown). Sequences 2, 3, and 4 share 99% sequence identity with sequence 1

AIM V培养基说明书

AIM-V® Medium CTS™Therapeutic Grade serum free cell expansion mediumGIBCO® AIM-V Medium CTS™ (Therapeutic Grade) is the first commercially available defined, serum-free formulation for proliferation and/or manipulation of T-cells and dendritic cells and manufactured in compliance with cGMP. AIM-V Medium CTS™ is an FDA 510(k) cleared device which is intended for human ex-vivo tissue & cell culture processing applications.Description Cat. No. SizeAIM-V® Medium CTS™, Liquid 0870112DK 1000mL AIM-V® Medium CTS™, Liquid 0870112BK 10L(Bag)IntendedUseFor human ex-vivo tissue & cell culture processing applications. CAUTION: When used as a medical device, Federal Law restricts this device to sale by or on the order of a physician. StorageStore medium at 2 to 8°C. Protect from light.Shelf Life14 monthsCulture Procedure:The procedure below serves as a general guideline for static T- cell and dendritic cell culture, regardless of vessel. For high-density culture in bioreactors, optimal procedures should be determined empirically by the investigator.T Cells Culture:1. Prepare fresh peripheral blood mononuclear cells (PBMCs)or rapidly thaw (< 1 minute) frozen vials of PBMCs cells in a 37°C water bath according to standard PBMC thawing protocols.2. Wash cells with DPBS CTS™ without calcium andmagnesium (Cat. No A12856), with 2-5% heat-inactivated human pooled Type AB serum according to the applications, if desired or required.3. Count cells using either electronic (i.e. Coulter Counter, Vi-Cell) or manual (i.e. hemocytometer) methods.4. Centrifuge cells and remove wash buffer.5. Resuspend PBMC at roughly 0.5-1x106 CD3+ T cells/mL inmedium supplemented with cytokines (e.g. IL-2), if used at culture initiation. Transfer the desired number of cells to the desired tissue culture vessel. A variety of protocols may be used for activating T-cells for subsequent expansion, including adding stimulatory antibodies or antigen presenting cells. Similarly, for either small or the large scale T-cell expansion, cells can be isolated, activated and expanded with Dynabeads® ClinExVivo™ CD3/CD28 or Dynabeads® CD3/CD28 CTS TM(Cat. No. 402-03D) according to instructions in the product insert. 6. Incubate the culture vessel at 37°C in a humidifiedatmosphere with 5% CO2. Feed and maintain cells at desired concentrations while cells are in log phase growth.To maintain log phase growth, it may be preferable to split cells to achieve a density of 0.5-1x106cells/mL whenever cell density gets above 1x106cells/mL (e.g. 2x106cells/mL, split 1:4 to continue culture at 0.5x106cells/mL). For optimal gas exchange in static plate cultures it is recommended that medium depth not exceed 1 to 1.2cm.Monocyte Derived Dendritic Cell Culture:1. Prepare fresh peripheral blood mononuclear cells (PBMCs).2. Plate PBMC in culture flask with 25 mL RPMI 1640 (Cat.No 72400) or AIM-V® Medium CTS™ (Therapeutic Grade).3. Incubate for 2 to 3 hours at 36 to 38˚C in a humidified atmosphere of 5% CO2 in air.4. Discard medium containing non-adherent cells.5. Wash the adherent cells (mainly CD14+ monocytes) threetimes with DPBS without calcium and magnesium (Cat. No A12856).6. Add medium containing 50 to 100 ng/mL recombinanthuman IL-4 (Cat. No. CTP0043 1mg or Cat. No. CTP0041 100ug) and 50 ng/mL recombinant human GM-CSF (Cat.No. CTP2011 100ug or Cat. No. CTP2013 1mg). Cell density should be between 1 to 3x105 cells/mL.7. Incubate cells at 36 to 38˚C in a humidified atmosphere of5% CO2 in air for 5 days. It is recommended to replace medium once after 3 days with fresh medium containing IL-4 and GM-CSF. Save all non-adherent or loosely adherentcells by centrifuging the removed culture medium 10 minutes at 200xg and adding the pellet to the fresh culture medium.8. After 6 days, the loosely adherent or non-adherent cellsshould display typical dendritic cell morphology and surface markers (CD1a, CD80, CD86, and HLA-DR).9. The maturation of dendritic cells is induced by the additionof either 1 µg/mL LPS or 50µl/mL TNF-α (cat. No. CTP3013 1mg or Cat. No. CTP3011 100ug) to the medium.Note: Alternatively to plastic adherence, monocytes can also be isolated by magnetic separation.Related ProductsDulbecco's Phosphate Buffered Saline CTS™ (DPBS) without calcium, magnesium (1X), liquid (A12856) L-Glutamine-200mM (100X), liquid (25030)Dynabeads ClinExVivo™ CD3/CD28 ®or Dynabeads CD3/CD28 ®CTS TM (402-03D) DynaMag™ CTS™ (121-02)IL-2 CTS™ REC HU (CTP0021 100ug or CTP0023 1mg) IL-7 CTS™ REC HU (CTP0071 100ug or CTP0073 1mg) IL-4 CTS™ REC HU (CTP0041 100ug or CTP0043 1mg) GM-CSF CTS™ REC HU (CTP2011 100ug or CTP2013 1mg) TNF-α CTS™ (CTP3011 100ug or CTP3013 1mg)Technical SupportFor additional product and technical information, such as Material Safety Data Sheets (MSDS), Certificate of Analysis,etc, please visit our website at /celltherapysupport/. For further assistance, please email our Technical Support team at celltherapysupport@The trademarks mentioned herein are the property of Life Technologies Corporation or their respective ownersReferences1.Rebecca J et al., (2010) Natural exposure to cutaneous anthrax gives long lasting T cell immunity encompassing infection-specific Epitopes. J. Immunol., 184: 3814 – 38212.Fabricius D et al., (2010) Prostaglandin E2 inhibits IFN-α secretion and Th1 costimulation by human plasmacytoid dendritic cells via E-prostanoid 2 and E-prostanoid 4 receptor engagement. J. Immunol., 184: 677 – 6843.Nesbit L et al., (2010) Polyfunctional T Lymphocytes Are in the Peripheral Blood of Donors Naturally Immune to Coccidioidomycosis and Are Not Induced by Dendritic Cells . Infect. Immun., 78: 309 - 3154. Jahrsdorfer B et al., (2010) Granzyme B produced by human plasmacytoid dendritic cells suppresses T-cell expansion . Blood, 115: 1156 – 11655.Csillag A et al., (2010) Pollen-Induced Oxidative Stress Influences Both Innate and Adaptive Immune Responses via Altering Dendritic Cell Functions. J. Immunol., 184: 2377 – 23856.Cornberg M et al., (2010) CD8 T Cell Cross-Reactivity Networks Mediate Heterologous Immunity in Human EBV and Murine Vaccinia Virus Infections. J. Immunol., 184: 2825 - 2838.7.Bellone S et al., (2009) Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8 Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8 CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines ++. J. Virol., 83: 6779 - 67898. Sato K et al., (2009) Impact of culture medium on the expansion of T cells for immunotherapy. Cytotherapy 11: 4-119.Liu ZW et al., (2009) A CD26-Controlled Cell Surface Cascade for Regulation of T Cell Motility and Chemokine Signals . J. Immunol ., 183: 3616 - 3624.10. Megyeri M et al., (2009) Complement Protease MASP-1 Activates HumanEndothelial Cells: PAR4 Activation Is a Link between Complement and Endothelial Function. J. Immunol., 183: 3409 - 3416. 11.Manfred L et al., (2005) Functional characterization of monocyte-derived dendritic cells generated under serum free culture conditions. Immunology letters 99: 209-21612. Nagorsen D et al., (2003) Biased epitope selection by recombinant vaccinia-virus (rVV)-infected mature or immature dendritic cells. Gene Therapy 10: 1754-1765 13. Lotem M et al., (2006) Presentation of tumor antigens by dendritic cells genetically modified with viral and nonviral vectors. J immunotherapy 29: 616-62714.Dietze B et al. (2008) An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: divergent maturation programs by IL-4 and LPS. Immunobiology 213:751–758.15.Meehan KR et al. (2008) Development of a clinical model for ex vivo expansion of multiple populations of effector cells for adoptive cellular therapy. Cytotherapy 10: 30–37.16. Ye Z et al. (2006) Human dendritic cells engineered to express alpha tumornecrosis factor maintain cellular maturation and T-cell stimulation capacity. Cancer Biother Radiopharm 21:613–622.17. Choi BH et al. (2006) Optimization of the concentration of autologous serum forgeneration of leukemic dendritic cells from acute myeloid leukemic cells for clinical immunotherapy. J Clin Apher 21:233–240. 18.Imataki O et al. (2006) Efficient ex vivo expansion of alpha24+ NKT cells derived from G-CSF-mobilized blood cells. J Immunother 29:320–327.19. Peng JC et al. (2005) Generation and maturation of dendritic cells for clinicalapplication under serum-free conditions. J Immunother 28:599–609. 20. Trickett AE et al. (2002) Ex vivo expansion of functional T lymphocytes from HIV infected individuals. J Immunol Methods 262:71–83.21.Carlens S et al. (2000) Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution. Exp Hematol 28:1137–1146.22.Kambe N et al. (2000) An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium. J Immunol Methods 240:101–110.23. Gerin PA et al. (1999) Production of retroviral vectors for gene therapy with thehuman packaging cell line FLYRD18. Biotechnol Prog 15:941–948. 24. Slunt JB et al. (1997) Human T-cell responses to Trichophyton tonsurans:inhibition using the serum free medium Aim-V. Clin Exp Allergy 27:1184–1192. 25. Kreuzfelder E (1996) Assessment of peripheral blood mononuclear cellproliferation by [2-3H]adenine uptake in the woodchuck model. Clin Immunol Immunopathol 78:223–227. 26.Causey AL (1994) A serum-free medium for human primary T lymphocyte culture. J Immunol Methods 175:115–121.27. Freedman RS et al. (1994) Large-scale expansion in interleukin-2 oftumorinfiltrating lymphocytes from patients with ovarian carcinoma for adoptive immunotherapy. J Immunol Methods 167:145–160. 28.Nomura K et al. (1993) [Study of adoptive immunotherapy for metastatic renal cell carcinoma with lymphokine-activated killer (LAK) cells and interleukin-2. II. Clinical evaluation.] Nippon Hinyokika Gakkai Zasshi 84:831–840. Japanese.29.Kaldjian EP et al. (1992) Enhancement of lymphocyte proliferation assays by use of serum-free medium. J Immunol Methods 147:189–195.30. Hayakawa K et al. (1991) Study of tumor-infiltrating lymphocytes for adoptivetherapy of renal cell carcinoma (RCC) and metastatic melanoma: sequential proliferation of cytotoxic natural killer and noncytotoxic T cells in RCC. J Immunother 10:313–325. 31.McVicar DW et al. (1991) A comparison of serum-free media for the support of in vitro mitogen-induced blastogenic expansion of cytolytic lymphocytes. Cytotechnology 6:105–113.32. Burg S et al. (1991) [Effect of different media on long-term cultivation of humansynovial macrophages.] Z Rheumatol 50:142–150. German. 33. Helinski EH et al. (1988) Long-term cultivation of functional human macrophagesin Teflon dishes with serum-free media. J Leukoc Biol 44:111–121. 34. Robyn S et al. (2007) RA8, A human anti-CD25 antibody against human Tregcells. Hybridoma 26:119–130. 35.Chena X et al. (2006) Induction of primary anti-HIV CD4 and CD8 T cell responses by dendritic cells transduced with self-inactivating lentiviral vectors. Cell Immunol 243:10–18.36. Grant R et al. (2008) CCL2 increases X4-tropic HIV-1 entry into resting CD4+ Tcells. J Biol Chem 283:30745–30753. 37.Hagihara M et al. (2003) Increased frequency of CD3/8/56-positive umbilical cord blood T lymphocytes after allo-priming in vitro. Ann Hematol 82:166–170.38. Wang Z et al. (2006) Application of serum-free culture medium for preparation ofA-NK cells. Cell Mol Immunol 3:391–395. 39. Morecki S et al. (1991) Retrovirus-mediated gene transfer into CD4+ and CD8+human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells. Cancer Immunol Immunother 32:342–352. 40.Johansen P et al. (2003) CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells. Eur J Immunol 34:91–97.June 2010Form No. 5047。

〈87〉 Biological Reactivity Tests, In Vitro

á87ñ BIOLOGICAL REACTIVITY TESTS, IN VITROThe following tests are designed to determine the biological reactivity of mammalian cell cultures following contact with the elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from the materials under test. It is essential that the tests be performed on the specified surface area. When the surface area of the specimen cannot be determined, use 0.1 g of elastomer or 0.2 g of plastic or other material for every mL of extraction fluid.Exercise care in the preparation of the materials to prevent contamination with microorganisms and other foreign matter. Three tests are described (i.e., the Agar Diffusion Tes t, the Direct Contact Tes t, and the Elution Tes t).1 The decision as to which type of test or the number of tests to be performed to assess the potential biological response of a specific sample or extract depends upon the material, the final product, and its intended use. Other factors that may also affect the suitability of a sample for a specific use are the polymeric composition; processing and cleaning procedures; contacting media; inks;adhesives; absorption, adsorption, and permeability of preservatives; and conditions of storage. Evaluation of such factors should be made by appropriate additional specific tests before determining that a product made from a specific material is suitable for its intended use. Materials that fail the in vitr o tests are candidates for the in viv o tests described in Biological Reactivity Tests, In Vivo á88ñ.PROCEDURES•T EST C ONTROLPositive control:Polyurethane film containing zinc diethyldithiocarbamate (ZDEC)2 or zinc dibutyldithiocarbamate (ZDBC) Cell culture preparation:Prepare multiple cultures of L-929 (ATCC cell line CCL 1, NCTC clone 929; alternative cell lines obtained from a standard repository may be used with suitable validation) mammalian fibroblast cells inserum-supplemented minimum essential medium having a seeding density of about 105 cells per mL. Incubate the cultures at 37 ± 1° in a humidified incubator for NLT 24 h in a 5 ± 1% carbon dioxide atmosphere until a monolayer, with greater than 80% confluence, is obtained. Examine the prepared cultures under a microscope to ensure uniform, near-confluent monolayers. [N OTE—The reproducibility of the in vitro biological reactivity tests depends upon obtaining uniform cell culture density.]Extraction solvents:Sodium Chloride Injectio n [see monograph—use Sodium Chloride Injectio n containing 0.9% of sodium chloride (NaCl)]. Alternatively, serum-free mammalian cell culture media or serum-supplemented mammalian cell culture media may be used. Serum supplementation is used when extraction is done at 37° for 24 h.•A PPARATUSAutoclave:Employ an autoclave capable of maintaining a temperature of 121 ± 2°, equipped with a thermometer, a pressure gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system that will allow for cooling of the test containers to about 20°, but not below 20°, immediately following the heating cycle. Oven:Use an oven, preferably a mechanical convection model, that will maintain operating temperatures in the range of 50°–70° within ±2°.Incubator:Use an incubator capable of maintaining a temperature of 37 ± 1° and a humidified atmosphere of 5 ± 1% carbon dioxide in air.Extraction containers:Use only containers, such as ampuls or screw-cap culture test tubes, or their equivalent, of Type I glass. If used, culture test tubes, or their equivalent, are closed with a screw cap having a suitable elastomeric liner. The exposed surface of the elastomeric liner is completely protected with an inert solid disk 50–75 µm in thickness. A suitable disk can be fabricated from polytef.Preparation of apparatus:Cleanse all glassware thoroughly with chromic acid cleansing mixture and, if necessary, with hot nitric acid followed by prolonged rinsing with Sterile Water for Injectio n. Sterilize and dry by a suitable process for containers and devices used for extraction, transfer, or administration of test material. If ethylene oxide is used as the sterilizing agent, allow NLT 48 h for complete degassing.•P ROCEDUREPreparation of sample for extracts:Prepare as directed in the Procedur e in á88ñ.Preparation of extracts:Prepare as directed for Preparation of extract s in á88ñ using either Sodium Chloride Injectio n [0.9% sodium chloride (NaCl)] or serum-free mammalian cell culture media as Extraction solvent s. [N OTE—If extraction is done at 37° for 24 h in an incubator, use cell culture media supplemented by serum. The extraction conditions should not in any instance cause physical changes, such as fusion or melting of the material pieces, other than a slight adherence.]•A GAR D IFFUSION T ESTThis test is designed for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens. Extracts of materials that are to be tested are applied to a piece of filter paper.Sample preparation:Use extracts prepared as directed, or use portions of the test specimens having flat surfaces NLT 100 mm2 in surface area.Positive control preparation:Proceed as directed for Sample preparatio n.Negative control preparation:Proceed as directed for Sample preparatio n.Procedure:Using 7 mL of cell suspension prepared as directed in Cell culture preparatio n, prepare the monolayers in plates having a 60-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with serum-supplemented culture medium containing NMT 2% of agar. [N OTE—The quality of the agar must be adequate to1Further details are given in the following publications of the American Society for Testing and Materials, 1916 Race St., Philadelphia, PA 19103: Standard test method for agar diffusion cell culture screening for cytotoxicity, ASTM Designation F 895-84; Standard practice for direct contact cell culture evaluation of materials for medical devices, ASTM Designation F 813-83.2ZDEC and ZDBC polyurethanes are available from the Food and Drug Safety Center, Hatano Research Institute, Ochiai 729–5, Hadanoshi, Kanagawa 257, Japan.support cell growth. The agar layer must be thin enough to permit diffusion of leached chemicals.] Place the flat surfaces of Sample preparatio n, Positive control preparatio n, and Negative control preparatio n or their extracts in an appropriate extracting medium, in duplicate cultures in contact with the solidified agar surface. Use no more than three specimens per prepared plate. Incubate all cultures for NLT 24 h at 37 ± 1°, preferably in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each sample, negative control, and positive control under a microscope, using a suitable stain, if desired.Interpretation of results:The biological reactivity (cellular degeneration and malformation) is described and rated on a scale of 0–4 (see Table 1). Measure the responses of the cell cultures to the Sample preparatio n, the Positive control preparatio n, and the Negative control preparatio n. The cell culture test system is suitable if the observed responses to the Negative control preparatio n is grade 0 (no reactivity) and to the Positive control preparatio n is at least grade 3 (moderate).The sample meets the requirements of the test if the response to the Sample preparatio n is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed.Table 1. Reactivity Grades for Agar Diffusion Test and Direct Contact TestGrade Reactivity Description of Reactivity Zone0None No detectable zone around or under specimen1Slight Some malformed or degenerated cells under specimen2Mild Zone limited to area under specimen and less than 0.45 cm beyond specimen3Moderate Zone extends 0.45–1.0 cm beyond specimen4Severe Zone extends greater than 1.0 cm beyond specimen•D IRECT C ONTACT T ESTThis test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very low- or high-density materials that could cause mechanical damage to the cells.Sample preparation:Use portions of the test specimen having flat surfaces NLT 100 mm2 in surface area.Positive control preparation:Proceed as directed for Sample preparatio n.Negative control preparation:Proceed as directed for Sample preparatio n.Procedure:Using 2 mL of cell suspension prepared as directed in Cell culture preparatio n, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the cultures, and replace it with 0.8 mL of fresh culture medium. Place a single Sample preparatio n, a Positive control preparatio n, and a Negative controlpreparatio n in each of the duplicate cultures. Incubate all cultures for NLT 24 h at 37 ± 1° in a humidified incubator containing 5 ± 1% of carbon dioxide. Examine each culture around each Sample preparatio n, a Positive controlpreparatio n, and a Negative control preparatio n, under a microscope, using a suitable stain, if desired.Interpretation of results:Proceed as directed for Interpretation of result s in Agar Diffusion Tes t. The sample meets the requirements of the test if the response to the Sample preparatio n is not greater than grade 2 (mildly reactive). Repeat the procedure if the suitability of the system is not confirmed.•E LUTION T ESTThis test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the specimens at physiological or nonphysiological temperatures for varying time intervals. It is appropriate for high-density materials and for dose-response evaluations.Sample preparation:Prepare as directed in Preparation of extract s, using either Sodium Chloride Injectio n [0.9% sodium chloride (NaCl)] or serum-free mammalian cell culture media as Extraction solvent s. If the size of the sample cannot be readily measured, a mass of NLT 0.1 g of elastomeric material or 0.2 g of plastic or polymeric material per mL of extraction medium may be used. Alternatively, use serum-supplemented mammalian cell culture media as the extracting medium to simulate more closely physiological conditions. Prepare the extracts by heating for 24 h in an incubator containing 5± 1% of carbon dioxide. Maintain the extraction temperature at 37 ± 1°, because higher temperatures may cause denaturation of serum proteins.Positive control preparation:Proceed as directed for Sample preparatio n.Negative control preparation:Proceed as directed for Sample preparatio n.Procedure:Using 2 mL of cell suspension prepared as directed in Cell culture preparatio n, prepare the monolayers in plates having a 35-mm diameter. Following incubation, aspirate the culture medium from the monolayers, and replace it with extracts of the Sample preparatio n, Positive control preparatio n, or Negative control preparatio n. The serum-supplemented and serum-free cell culture media extracts are tested in duplicate without dilution (100%). The Sodium Chloride Injectio n extract is diluted with serum-supplemented cell culture medium and tested in duplicate at 25% extract concentration.Incubate all cultures for 48 h at 37 ± 1° in a humidified incubator preferably containing 5 ± 1% of carbon dioxide. Examine each culture at 48 h, under a microscope, using a suitable stain, if desired.Interpretation of results:Proceed as directed for Interpretation of result s in Agar Diffusion Tes t but use Table 2. The sample meets the requirements of the test if the response to the Sample preparatio n is not greater than grade 2 (mildly reactive).Repeat the procedure if the suitability of the system is not confirmed. For dose-response evaluations, repeat the procedure, using quantitative dilutions of the sample extract.1Slight Less than or equal to 20% of the cells are round, loosely attached, and without intracytoplasmic granules; occasional lysed cells are present 2Mild Greater than 20% to less than or equal to 50% of the cells are round and devoid of intracyto-plasmic granules; no extensive cell lysis and empty areas between cells 3Moderate Greater than 50% to less than 70% of the cell layers contain rounded cells or are lysed 4Severe Nearly complete destruction of the cell layers ADDITIONAL REQUIREMENTS •USP R EFERENCE S TANDARDS á11ñUSP High-Density Polyethylene RS (Negative Control)Table 2. Reactivity Grades for Elution TestGradeReactivity Conditions of All Cultures 0None Discrete intracytoplasmic granules; no cell lysis。

sirius red染色原理

sirius red染色原理sirius red染色是一种常用于组织切片中胶原纤维可视化的染色方法。

它是一种酸性染料，能够专一地结合胶原纤维，使其显色。

在sirius red染色中，胶原纤维会变成红色至红紫色。

以下是sirius red染色的原理及其应用的解释。

1.原理：sirius red染色基于酸性染料与胶原纤维之间的亲和性。

胶原蛋白是一种主要组成结缔组织的蛋白质，具有重要的功能，例如提供机械支持和促进伤口愈合。

在sirius red染色中，胶原纤维中的阳离子与酸性染料中的负离子相互吸引形成络合物，并产生显色。

2.染色过程：sirius red染色的步骤相对简单。

首先，切片必须准备好，通常是经过固定、脱水和包埋后制备的石蜡切片。

然后，将切片浸泡在硝酸银溶液中，以消除切片中的镍离子。

接下来，将切片浸泡在sirius red染料溶液中，通常为sirius red F3BA溶液。

染料渗透到切片中的胶原纤维中，并与其形成络合物。

最后，切片通过漂洗和脱水等程序处理，并用玻璃封片封装。

3.应用：sirius red染色主要应用于研究和诊断领域。

它在病理学中的应用非常广泛，可以用于评估或诊断多种疾病，例如纤维化、肝纤维化、心肌纤维化、肺纤维化等。

通过sirius red染色，可以观察和定量分析组织中胶原纤维的分布和丰度，从而了解疾病的病理变化。

此外，sirius red染色还可用于研究胶原纤维在生理和病理条件下的重建和变化，以及在创伤愈合、组织工程和生物材料研究等领域的应用。

总之，sirius red染色是一种可靠的方法，主要用于胶原纤维的可视化。

通过该染色方法，我们可以了解到胶原纤维在不同的疾病和生理条件下的变化，从而为疾病的诊断和治疗提供参考和依据。

由于其简单性和高效性，sirius red染色被广泛应用于医学和科学研究领域。

cell culture basics by invitrogen

• • • • •
a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals) 生长因子 hormones gases (O2, CO2) a regulated physico-chemical environment (pH, osmotic pressure, temperature)
Primary culture refers to the stage a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the
passaged) by transferring them to a population. new vessel with fresh growth medium to provide more room for continued growth.
Finite vs Continuous Cell Line
Cell Culture Laboratory Safety
Share | In addition to the safety risks common to most everyday workplaces such as electrical and fire hazards, a cell culture laboratory has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive, or mutagenic solvents and reagents. Common hazards are accidental punctures with syringe needles or other contaminated sharps, spills and splashes onto skin and mucous membranes, ingestion through mouth pipetting, and inhalation exposures to infectious aerosols.

人肿瘤坏死因子α（TNF-α）英文说明书

Human TNF-αFOR RESEARCH USE ONLYAssay range：：：：20 ng/L -400 ng/L 96determinationsPurposeThis kit allows for the determination of TNF-α concentrations in Human serum, cell culture supernates and other biological fluidsPrinciple of the assayThe kit assay Human TNF-α level in the sample, use Purified Human TNF-α antibody to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combined TNF-α antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution, TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard（800ng/L）0.5ml×1 bottle3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle4 Sample diluent 6ml×1 bottle 10 Instruction 15 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane26 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevantliterature, and should be experiment as soon as possible after the extraction. If it can’t,specimen can be kept in -20 ℃to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:400 ng/L 5 Standard 150µl Original density Standard+150µl Standard diluent200 ng/L 4 Standard 150µl 5 Standard+150µl Standard diluent100 ng/L 3 Standard 150µl 4 Standard+150µl Standard diluent50 ng/L 2 Standard 150µl 3 Standard +150µl Standard diluent25 ng/L 1 Standard 150µl 2 Standard +150µl Standard diluent2.add sample: Set blank wells separately (blank comparison wells don’t add sample andHRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40µl to testing sample well, then add testing sample 10µl (sample final dilution is5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5.Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50µl to each well, except blank well.7. Incubate: Operation with 3.8. Washing: Operation with 5.9.Color: Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃10.Stop the reaction：Add Stop Solution50µl to each well, Stop the reaction(the blue colorchange to yellow color).11. Assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.。

《科技英语》课后习题答案

MainContent:UNIT1MATHEMATICSI.TextOrganizationParts ParagraphsMainIdeasPartOne Paras.1-3 Gametheorycanbedefinedasthescienceofstrategywhichstudiesbothpureconflicts(zero-sumgames)andconflictsincooperativeforms.PartTwo Paras.4-11 Therearetwodistincttypesofstrategicinterdep endence:sequential-movegameandsimultaneous-movegame.PartThre e Paras.12-19Thetypicalexamplesofgametheoryaregivenasthebasicprinciplessuchasprisoners’dilemma,mixingmoves,strategicmoves,bargaining,concealingandrevealinginformation.PartFour Para.20 Theresearchofgametheoryhassucceededinillustratingstrategiesinsituationsofconflictandcooperationanditwillfocusonthedesignofsuccessfulstrategyinfuture.nguagePointsThegamesitstudiesrangefromchesstochildrearingandfromtennistotak eovers.(Para.1)Paraphrase:Thegamesit(gametheory)studiesextendsfromchesstochild bringing-upandfromtennistohandovers.range:v.tovarybetweenlimits,extend,runinalinee.g.(1)Thepricerangesfrom$30to$80.(2)Theboundaryrangesfromnorth tosouth.takeover:n.theactoraninstanceofassumingcontrolormanagementoforr esponsibilityforsth.接收、接管e.g.TheeconomyofHongkonggoeswellafteritstakeover. GametheorywaspioneeredbyPrincetonmathematicianJohnvonNeumann.(P ara.2)pioneer:v.tobeapioneer;tooriginate(courseofactionetc.,followedl aterbyothers)e.g.Thenewtreatmentforcancerwaspioneeredbytheexpertsofstatehosp ital.pioneer:n.originalinvestigatorofsubjectorexplorerorsettler;init iatorofenterprisee.g.Theyounggenerationwasgreatlymotivatedbythepioneers’exploit s.Thatis,theparticipantsweresupposedtochooseandimplementtheiracti onsjointly.(Para.2)Paraphrase:Thatis,theplayerswereexpectedtoselectandcarryoutthei ractionstogether. …hemustanticipateandovercomeresistancetohisplans.(Para.3) anticipate:v.1)toexpectorrealizebeforehand;toforeseee.g.Theexpertsareanticipatingthenegativeeffectsofairpollution. anticipate:v.2)todealwithorusebeforepropertime预支e.g.Tedwasnotusedtosavingmonthlyandhewouldalwaysanticipatehisin come. Theessenceofagameistheinterdependenceofplayerstrategies.(Para.4 )Paraphrase:Thekeyprincipalofagameisthatplayerstrategiesaredepen dentoneachother.essence:n.1)thequalitywhichmakesathingwhatitis;theinnernatureor mostimportantqualityofathinge.g.Thetwothingsarethesameinoutwardformbutdifferentinessence. essence:n.2)extractobtainedfromasubstancebytakingoutasmuchofthe massaspossiblekessence;essenceofpeppermint(椒薄荷、椒薄荷油) interdependence:n.thequalityorfactofdependingoneachotherinter-为前缀，意为betweeneachother,类似的词还有interchange、intermarry、international、interview等。

普病知识100问(1)-1

普病知识100问1.什么是植物病害？植物病害对农业生产都有哪些影响？植物在自然界中受到有害生物或不良的环境条件的影响，使其正常的生长发育受阻，在代谢上发生了改变，造成了损伤或损害叫植物病害。

影响：1.降低产量2.降低品质3.产生有毒物质，使人畜中毒4.限制了农作物的栽培5.影响农产品的运输和贮藏2.1845年在哪个地区发生了一个重大植物病害对人类产生了什么影响？爱尔兰饥谨：1845年马铃薯晚疫病在爱尔兰大流行，饿死几十万人、迫使150 万人移居美国。

植物病理学由此诞生。

3.什么是植物病理学？植物病理学之父是谁？研究植物病害的发病原因、病害发生发展规律、植物与有害生物间的相互作用机制以及病害预测及其防治的学科。

德巴利（De Bary）是植物病理学的奠基人，被尊称为植物病理学之父。

4.植物病害类型有哪些？如何认识植物病害？非侵染性病害，侵染性病害5.什么是病症？包括几种类型？病症：病原物在植物体上表现出来的特征性结构.类型：霉状物、粉状物、小黑点、菌核、菌脓6.什么是病状？包括几种类型？病状：是指发病植物本身所表现出来的反常现象。

类型：变色、坏死、萎蔫、腐烂、畸形7.什么是侵染性病害？什么是非侵染性病害？由于生长环境条件不合适，物理或化学因素造成的，是非侵染性的，这不能传染的病害叫非侵染性病害。

有时叫生理病害。

由于病原物对植物侵染造成的，因可传染，又叫传染性病害。

8.什么是病原物？病原物主要有哪些类群？对寄主具有致病性的寄生物叫病原物。

类群：9.什么是典型症状？什么是隐症现象？典型症状(typical symptom)：一种病害在不同阶段或不同抗病性的品种上或者在不同的环境条件下出现不同的症状，其中一种常见症状成为该病害的典型症状。

隐症现象(masking of symptom)：病害症状出现后，由于环境条件的改变，或者使用农药治疗后，原有症状逐渐减退直至消失。

10.什么是寄生性？什么是致病性？寄生物获得营养的能力叫寄生性。

sf9细胞污染

细胞培养中的黑胶虫污染摘要：预防和避免污染是细胞培养成功的关键，黑胶虫（也称黑焦虫）是近几年来几乎出现在每个细胞培养实验室。

但是现在无法对黑胶虫的确认和鉴别，导致学术界说法不一，笔者收集了关于黑胶虫的资料，对黑胶虫进行类系统描述，对黑胶虫出现的情况对细胞培养的影响以及黑胶虫污染后的有效处理进行介绍。

关键词：黑胶虫污染处理细胞培养The pollution of black dots in cell culture(Grade 2005, Biotechnology, School of Life Science and Engineering)Abstract Pollution prevention is the key to success in cell culture, black dots (also called black particles) almost appeared in every cell culture laboratory in past few years .But now no one able to confirm and identify what the black dots is. This lead to appear different academic arguments.The author collected information about the black dots to make a description about black spots which similar to the systemic description,and Introduce when aand where the black dots come out. At the same time give some advice to deal with the black dots.Key words Pollution black dots cell culture前言污染是细胞培养的大敌。

细胞培养(英文版本)

• 1927: Carrel and Rivera produced the first viral vaccine Vaccinia.
• 1933: Gey developed the roller tube technique
Contd..
• 1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture.
• 1954: Abercrombie observed contact inhibition: motility of diploid cells in monolayer culture ceases when contact is made with adjacent cells.
• 1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.
• Cell culture was first successfully undertaken by Ross Harrison in 1907
• Roux in 1885 for the first time maintained embryonic chick cells in a cell culture
connective tissue in serum • and plasma. • 1903: Jolly observed cell division of salamander leucocytes in vitro. • 1907: Harrison cultivated frog nerve cells in a lymph clot held by the

蒋悟生生物学专业英语(第四版) 第1课-Inside the Living Cell

• Centriole ['sentrɪəʊl]细胞中心粒，中心体; (see centre)
• Chemotaxis 趋化作用，趋向特征，指人体细胞，细菌，单细胞或多细胞生物在他们所处的环境中的某些化学物质的指令下，进行定向运动的特征。 chemo- + Greek taxis "arrangement”
几个核糖体可能附着在一条mrna链上；这种组合被称为多聚体。大多数细胞蛋白质是在细胞质中的核糖体上制造（合成）的。可输出蛋白和膜蛋白的合成通常（是核糖体）与内质网联合（完成的）。
作者：momoTian
The endoplasmic reticulum, a lacy array of membranous sacs, tubules, and vesicles, may be either rough (RER) or smooth (SER). Both types play roles in the synthesis and transport of proteins. The RER, which is studded with polysomes, also seems to be the source of the nuclear envelope after a cell divides.
the genetic material(DNA) on chromosomes. (In prokaryotes the hereditary material is found in the nucleoid.)
The nucleus also contains one or two organelles-the nucleolithat play a role in cell division.

细胞培养中形成的条状物

细胞培养中形成的条状物英文回答：Cell culture is a widely used technique in biological research, where cells are grown and maintained in a controlled environment outside of their natural context. Sometimes, during the process of cell culture, cells can form elongated structures known as "stripes" or "bands". These structures can be observed under a microscope and are often a result of cell migration or alignment.There are several reasons why cells may form stripes in cell culture. One possibility is that the cells are responding to a gradient or directional cue in the culture environment. For example, if there is a concentration gradient of a particular growth factor or signaling molecule in the culture medium, cells may migrate towards the higher concentration and align themselves in a stripe-like pattern.Another possibility is that the cells are responding to physical cues, such as the surface topography or stiffnessof the culture substrate. Cells have the ability to sense and respond to their mechanical environment, and certain patterns or textures on the culture surface can influence cell behavior. For instance, if the culture substrate has parallel grooves or ridges, cells may align along these features and form stripes.In addition, cell-cell interactions can also play arole in stripe formation. Cells communicate with each other through various signaling molecules, and these interactions can influence their behavior and organization. For example, if certain cells in the culture secrete a signalingmolecule that attracts or repels neighboring cells, thiscan result in the formation of stripes.To illustrate these concepts, let's consider an example. Imagine we are culturing fibroblast cells on a substratewith parallel grooves. As the fibroblasts migrate and divide, they sense the grooves on the substrate and align themselves along the direction of the grooves. Over time,this alignment leads to the formation of stripes of fibroblast cells. This phenomenon can be observed under a microscope and provides valuable insights into how cells respond to their physical environment.中文回答：细胞培养是生物研究中广泛使用的技术，通过在受控环境中培养和维持细胞，使其在其自然环境之外生长。

欧洲药典7.5版

EUROPEAN PHARMACOPOEIA 7.5
INDEX
To aid users the index includes a reference to the supplement in which the latest version of a text can be found. For example : Amikacin sulfate...............................................7.5-4579 means the monograph Amikacin sulfate can be found on page 4579 of Supplement 7.5. Note that where no reference to a supplement is made, the text can be found in the principal volume.
English index ........................................................................ 4707
Latin index ................................................................................. 4739
EUROPEAN PHARMACOPபைடு நூலகம்EIA 7.5
Index
Numerics 1. General notices ................................................................... 7.5-4453 2.1.1. Droppers...................