Nunc nunclon products are tested with two cell lines l929, hel 299 or f2002 and one primary chick embryo cell culture for monolayer formation, plus cell line v794 for cloning efficiency. This chapter outlines protocols used to isolate and culture murine bone marrowderived and peritoneal macrophages. Cell concentration series for the optimization of cell confluence. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. Resistance can be circumvented by modulating agents such as verapamil and quinine. Clone 929 was established by the capillary technique for single cell isolation from the 95th subculture generation of the parent strain. Determine the number of cells required for each product by multiplying the plating density by the surface area. Can anyone advise me on using l929 cells to produce. Growing l929 cells and collecting conditioned media. Balbc 3t3 cells are routinely grown as a monolayer in tissue culture grade flasks e. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e.
Every step of the way, a wide range of cell health products maintaining healthy cells is the key to experimental success and reproducible research results. Pdf differentiation capacity of mouse l929 fibroblastic cell line. Thermo scientific nunc nunclon cell culture products are tested for cell growth and plating efficiency using several different cell lines. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture counting cells in a hemocytometer. Protocol for hela cell culture and plasmid transfection av. Culturing l929 cell line on a thermo scientific nunc nunclon cell. How to count and calculate the number of cells from a stock flask or culture dish counting cells in a countess ii. Under these conditions, the bone marrow monocytemacrophage progenitors will proliferate and differentiate into a homogenous population of mature bmms. The cells were cultured in dmem supplemented with 10% fcs and penicillinstreptomycin. This information is also available as a pdf from our main menu, under customer support click here. The l strain was derived from normal subcutaneous areolar and adipose tissue of a 100 day old male c3han mouse. Ecacc general collection catalogue no 85103115 cell line name. Thaw l929 fibroblasts stored in 140c in a 37c degree water bath for 2 minutes.
The cell treatment protocol was performed with the tested solutions at doses of 0. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell. L929 mouse, subcutaneous connective tissue, a549 human caucasian lung carcinoma, nclh95 human lung adenocarcinoma, 22rv1 human prostate carcinoma and raw 264. The l strain was derived from normal subcutaneous areolar and adipose tissue of a 100 day old. Protocol for reverse transfection of adherent and suspension cells in a 24well plate note. The culture medium was then discarded and replaced with 20 ml of fresh medium and the conditioned medium was collected 72 h later. C 0 genjet in vitro dna transfection reagent for l929 cell.
Mammalian cell tissue culture techniques protocol abcam. Test method protocol for balbc 3t3 neutral red uptake cytotoxicity assay phase iii validation study november 4, 2003. Test method protocol for the balbc 3t3 neutral red uptake. Scaleup quantities and volumes according to the number of cellswells to be transfected see table 1. Induction of apoptosis in tnftreated l929 cells in the. L929, l 929, nctc, l929nctc, nctc l929 mouse c3han areolar and adipose tissue the parent l strain was derived from normal subcutaneous areolar and adipose tissue of a 100dayold male c3han mouse by wr earle in 1940. Effects of calcium hypochlorite and araraquara school of. As mmp9 and mmp2 in the culture supernatants of l929 were previously reported, this was served as reference for mmp2 and mmp9 activity. This tech note describes a procedure for culturing l929 cell line on a nunclon treated surface. The product sheet that came with the cells has little information. Similar or standard freezing protocols may be substituted. Gas the culture and incubate in warm room overnight. Cytotoxicity evaluation of endodontic pins on l929 cell line.
The cells were maintained at 37 c in a humidified 5% co 2 incubator nbs eppendorf, germany using standard cell culture procedures. Cell applications recommends the user follow the steps. Cell culture ht29 cells were cultured in mccoys 5a culture medium invitrogen. Examine and make sure the cell culture is free of contamination, in healthy situation and proper confluency, etc. In this protocol, bone marrow cells are grown in culture dishes in the presence of mcsf, which is secreted by l929 cells and is. Can anyone advise me on using l929 cells to produce culture. How to count and calculate the number of cells using an automated cell counter. Seed the l929 cells in the plate 24 well with the concentrations indicated in figure 2. If use of this culture results in a scientific publication, it should be cited in the publication as. One of the first to be established in continuous culture. The parent l strain was derived from normal subcutaneous areolar and adipose tissue of a 100dayold male c3han mouse by wr earle in 1940.
Ecacc general collection catalogue no 85011425 cell line name. Cell growth curves for different cell lines and their. In vitro cytotoxicity studies of surface modified cas. Maintaining healthy cells is the key to experimental success and reproducible research results. This is a robust 20 day differentiation protocol to direct mouse embryonic stem cells mescs towards the macrophage lineage. Cell culture protocols thermo fisher scientific jp. Tissue culture flasks incubator trypsin invitrogen falcon tubes 0. Lift cells with 5 ml 1x trypsinedtapbs, centrifuge as before, resuspend in 15 ml dmem10 and transfer to a t75 tissue culture flask. This indicates that in a migrating l929 fibroblast actin fibres span as a scaffold across the cell. A confluent monolayer is an adherent cell culture dish, plate or flask in which the cells have formed a single layer over the entire surface area available for growth. Nctc clone 929 clone of strain l also known as l929 was subsequently derived in march 1948.
Two technical replicates of each concentration and surface are performed. Strain l was one of the first cell strains to be established in continuous culture, and clone 929 was the first cloned strain developed. I ve got some l929 cells that have been in dmem with only lglu, no antibiotics. To give you confidence in the health of your cells every step of the way, weve highlighted the technologies and products within cell biology that are critical to maintaining optimal cell health. Supernatant from l929 is used in macrophage cultures as a source of macrophagecolony stimulating factor mcsf 17, 18. Pellet 3 ml of mycobacterial cultures at 2400g for 10 min. L929, l 929 mouse c3han connective tissue the parent l strain was derived from normal subcutaneous areolar and adipose tissue of a 100dayold male c3han mouse by wr earle in 1940.
Cells were then washed twice in optimem invitrogen, carlsbad and stimulated. Three technical replicates are performed for each surface. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Strain l was one of the first cell strains to be established in continuous culture. Fibroblast cell culture protocols the progeria research. Furthermore, we describe how these macrophages can be polarized to obtain specific macrophage subsets with special relevance to atherosclerosis. For activation, cells were exposed to various concentrations of lps, polyi. The capacity for a t cell to secrete certain cytokines can be measured using an intracellular cytokine staining protocol. The ends of the scaffold in the cell front contact the substrate more tightly fak content. Also, a method to deliver dna or small interfering rnas sirnas into these hardtotransfect cells is described. Perform a cell count to determine the viability of cells 3.
Resuspend cultures in 3 ml fresh 7h9c media2 alternatively larger culture volumes can be pelleted and concentrated into 3 ml. Culture and characterization of microglia from the adult. Cell culture cells were plated in sixwell culture plates 3 106well overnight. How to count and calculate the number of cells from a stock flask or culture dish.
Incubate the cells for 24 hours at 37c under standard cell culture conditions. The local fak accumulation artifact in the cells outmost rear is a regulation failure. Macrophages display plasticity in both phenotype and function. Cell culture guidelines the following is a general guideline for culturing of cell lines. Cell counting kit8 product description cell counting kit8 is a colorimetric assay for the determination of viable cell numbers and can be used for cell proliferation assays as well as cytotoxicity assays. For optimum results, cells for cryopreservation should be in log phase of growth. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. This adriamycinresistant cell line has been developed by exposure of the parent l929 murine fibroblast cell line ecacc catalogue number 85011425 to increasing concentrations of doxorubicin invitro. Strain l was one of the first cell strains to be established in continuous culture, and clone 929 was the first. Pipette thawed l929 gently into a 50 ml falcon tube containing 20 ml of warm dmem 10. Mcsf promotes survival of macrophages and induces the. The following is a general guideline for culturing of cell lines.
Mouse c3han connective tissue cell line description. Animal cell culture guide tips and techniques for continuous cell lines 1. This cell line is neither produced nor fully characterized by atcc. L929 cell line from mouse 85011425, mouse c3han connective. Please read the important technical information that follows before handling ecaccsupplied cell lines. Pipette into at t25 tissue culture flask, incubate at 37c, 5% co.
Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Some reagents and supplements will require filter sterilization if they are not provided sterile. Nctc clone 929 l cell, l929, derivative of strain l. Cells are harvested from the lungs and incubated with anticd3. Compared to immortalized cell lines, however, primary cells can be finicky and present challenges during culture unless proper care is taken. The aim of this study was to evaluate the cytotoxic potential of a type of endodontic pin on l929 cell line according to the uni en iso 109932009 rule. In this protocol, bone marrow cells are grown in culture dishes in the presence of mcsf, which is secreted by l929 cells and is used in the form of l929 conditioned medium. The optimal transfection conditions for l929 cell line as well as a general starting point for optimization are given in the standard protocol described below. Centrifuge the cell suspension at 1500 rpm for 5 minutes.
Gelatinzymography revealed the presence of both mmp2 and mmp9 in the culture supernatants of dla and l929 cancer cell lines. A compilation of cell biology related application notes, technical guides, bulletins, videos, and more available for downloading and viewing. The protocol below describes how to subculture adherent and suspension cultured cells. The protocol is optimized for transfection in a 24well plate format. Nctc clone 929 l cell, l 929, derivative of strain l atcc ccl1. I just tried out your method and was just wondering how you calculate your %. The protocol has been scaled up to be used with the plate 24 well. Mar 20, 2015 compared to immortalized cell lines, however, primary cells can be finicky and present challenges during culture unless proper care is taken. The following protocol is given for transfection in 24well plates, refer to table 1 for transfection in other culture formats. General details of cell culturing and subculturing.
Jun 04, 20 protocol 1 dna extraction part 1 duration. Incubate cells in a 37c incubator with 5% co 2 for four days. L929 cells were maintained in dulbeccos modified eagle medium dmem wako, osaka, japan supplemented with 10% heatinactivated fetal bovine serum and penicillin 100 umlstreptomycin 100. Then i wash the plates on day 4 to get rid of other cells ad fresh media dmem, 10% low endotoxin fbs, gln, ab, 50nm.
Cell culture basics handbook thermo fisher scientific. For best results, start with 1g of dna and 2l of transfection reagent. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. The l929 cells were grown in 75 cm 2 cell culture flasks cellstar, greiner bioone, gloucestershire, uk and were allowed to reach 90% confluency. Description this adriamycinresistant cell line has been developed by exposure of the parent l929 murine fibroblast cell line ecacc catalogue number 85011425 to increasing concentrations of doxorubicin invitro l929a cells can be used in the development of novel anticancer treatments. Dilute cells into growth medium and seed cell culture product. Recently i purchased l929 cells from atcc to condition media for macrophage differentiation. L929 fibroblast cells were procured from national centre for cell sciences nccs, pune, india and grown in dulbeccos modified eagles medium, dmem sigma aldrich, usa. Confluency confluency is a marker for when to subculture your cells.
Intracellular cytokine staining an overview sciencedirect. We do not guarantee that it will maintain a specific morphology, purity, or any other property upon passage. L929a cells can be used in the development of novel anticancer treatments. Subclone of parental strain l, established by w r earle in 1940.
122 636 1418 75 10 1254 1204 1074 562 1423 1163 335 917 745 709 1499 947 1618 632 1383 1362 480 474 1022 610 408 467 674 887 995 175 621 294 198 34