Bv2 cell culture protocol pdf

Primary cultures of hypothalamic neurons treated with culture medium from lpstreated bv2 cells increased crf, crfr, pcreb and camp levels compared to untreated neurons. The suitability of bv2 cells as alternative model system. It includes a presentation of equipment needed, cell culture medium and supplements, cell culture monitoring, and a protocol describing the steps for subculturing of microglia cell lines. With the bv2 cell line, there appears to be a tradeoff. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. All culture incubations should be performed in a humidi. What might be the optimum cell density for bv2 cells for 5 days culture. Dennis selkoe harvard university and cultured in dmem with 10% fbs and 1% penicillinstreptomycin. Jun 12, 20 cell culturing of human and murine microglia cell lines will be described in this chapter. Lpsinduced prongf synthesis and release in the n9 and bv2. The microglial cell line, bv2, is cultured every 34 days in new medium in a t75 flask. Chart showing surface area, seeding density, cells at confluency, and volumes of versene, trypsin and medium for various culture dishes, plates and flasks.

This article offers general methodological guidelines in order to set up an experiment in view of evaluating cellular changes mediated by secreted soluble factors using an insert co culture system. Il4polarized bv2 microglia cells promote angiogenesis by. However, some cells, particularly primary cells, will require growth on special. Immunocytochemistry was performed as described previously. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Mouse embryonic stem cell culturing protocols 4 of 6 form 105 rev b072214 7. Once cells are detached, add back 510 ml media and transfer to centrifuge tube 15ml sterile centrifuge tube. The confluent bv2 cells were plated in sixwell plates with polyllysine coated cover slips at a density of 1. Glucose transporter 1 critically controls microglial. Over the years a number of different microglial cell culturing. Mycoplasmal cell culture contamination occurs due to. Note that cell culture conditions vary for each cell type.

These t75 flasks work great for the culturing of bv2 cells. As there is no in vitro method available yet that recapitulates all. Development of an insert co culture system of two cellular types in the absence of cell cell contact. Cyclophilin isomerase activity was confirmed by the kofron method. Bv2 cell culture and polarization mouse microglial cell line bv2 cells were cultured in a complete medium roswell park memorial institute rpmi 1640 with lglutamine, supplemented with 10% fe tal bovine serum fbs, 100 uml penicillin and 100.

Ensure that incubators, cabinets, centrifuges and microscopes are cleaned and serviced at regular intervals. Cell line and culture conditions murine microglial bv2 cells, originally developed by dr v bocchini at university of perugia perugia, italy. Cell line authentication str profiling aids in the detection of misidentified, crosscontaminated, or genetically drifted cells, which invalidate research results. Can anyone advise me on how to maintain bv 2 cell line. Bestbio assay according to the manufacturers protocol.

Synthesis and upregulation of prongf protein significantly occurred in n9 and bv2 cells 12h48h after lps exposure, whereas no significant changes of probdnf and mmp9 were observed in these microglial cell lines with lps insult. The suitability of bv2 cells as alternative model system for primary. Bv2 cells were plated in six well plates in 10% fbs culture medium and allowed to reach 80% confluence. At 4 h, 8 h and 12 h after the low glucose and hypoxia treatments, the cell viability in each group was. The bv2 cell line was generated by transduction of neonatal primary. Total rna was prepared from bv2 cells by using the trizol reagent invitrogen corporation according to the manufacturers protocol.

Preliminary studies indicated that the solvent had no effect on cell viability at the concentration used. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation. The immortalised murine microglial cell line bv2 has been usedfrequently as a substitute for. The suitability of bv2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation anja henn, s. Optimization of concentration or incubation condition of the primary antibody and the secondary antibody for your own specimen is necessary. Resuspend the cell pellet in r10 and count viable cells using a hemocytometer and trypan blue. Bv2 mouse microglial cells were maintained in dulbeccos modified eagle medium. Materials and methodsby in vitro culture of n9 and bv2 cell lines and lipopolysaccharide lps stimulation model, generation and release of prongf, probdnf and mmp9 was studied in the activated microglial cells by immunocytochemistry, western blotting and bioassay methods. Antineuroinflammatory and neuroprotective effects of. Immunocytochemistry and immunofluorescence protocol. The immortalised murine microglial cell line bv2 has been used frequently.

Cell culturing of human and murine microglia cell lines. Detailed protocols for most of these basic procedures are already published freshney, 2005 and will not be presented here. Bv2 cell culture, primary cultures of microglia and hypothalamic neuron culture. Cell viability was determined by measuring the absorbance at 490 nm after addition of the mts reagent, and the results are expressed as the percentage of surviving cells over control cells without pmba treatment. Bv2 cells were plated overnight in 12well plates at a density of 5. Bv2 cells were cultured in a sixwell plate and infected with adklf2 moi 10 or transfected with siklf2 for 24 h. Protocol overview the bv2 cells are cultured at roughly 12 million cells in a t75 flask in 10 mls of dmem containing 10% of heat inactivated bovine serum and 2% of supplement. The sirna complex suspension was added dropwise to the 24well cell culture plate. Thank you for your understanding, patience and flexibility as atcc does everything it can to help reduce the impact of. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. Bv2 microglia and pc12 cells were purchased from the institute of basic medical sciences of the china science academy. Treatments of cck solution, incubation time, and analysis methods were performed according to a previous study, with some modifications 35. Oligonucleotide microarray analysis of apoptosis induced by. The bv2 cells were assessed for microglia cell markers and tested 90%.

Dmem supplemented with 10% fetal bovine serum fbs and 1% penicillinstreptomycinneomycin psn to 37 c. I have to treat the bv2 cells repeatedly for 5 days daily treatment with an immunomodulator. In vitro examination of microglianeuron crosstalk with bv2. Protocol mycoplasma detection test harvard university. The bv2 cells express the nuclear vmyc and the cytoplasmic vraf oncogene products as well as the env gp70 antigen at the surface level. The bv2 cell line produces an enveloped recombinant ecotropic retrovirus capable of infecting murine cells only. Activation of bv2 microglia by lipopolysaccharide triggers. The viability should be over 90% to ensure the cells are healthy. The cells were treated with different concentrations of dha 0, 10, 20, 40 and 80 m for 24 h, and the cck8 solution was added to each. Wash cells briefly with hanks solution prior trypsin treatment note. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other betalactam antibiotics that target cell wall synthesis. Rna sequencing analysis reveals quiescent microglia isolation. Briefly, grouped bv2 cells were seeded in a 96well plate and cultured for 24 h.

Autophagy down regulates proinflammatory mediators in bv2 microglial cells and rescues both lps and alphasynuclein induced neuronal cell death skip to main content thank you for visiting. When using accutase, cells can be plated directly into culture vessel without centrifuging to pellet cells as long as accutase is inactivated by the addition of the. In vitro examination of microglianeuron crosstalk with. Activation of bv2 microglia by lipopolysaccharide triggers an. The method of sedimentation was used to study the repair of sarcoma 37 cell nucleoid after irradiation of cells in vitro or after irradiation of mice with a dose of 9. Development of an insert coculture system of two cellular. Cells were treated with various concentrations of pmba for 24 h. Cell damage produced by magnetic fluid hyperthermia on. The bv2 cells were assessed for microglia cell markers and tested 90% positive for nonspecific esterase activity and all lacked peroxidase activity. Cystatin f involvement in adenosine a 2a receptormediated. Place the lid on the plate and incubate according to previously established cell culture protocols. Some reagents and supplements will require filter sterilization if they are not provided sterile. Klf2 protects bv2 microglial cells against oxygen and glucose.

Murine microglial cell line bv2 cells kindly provided by katerina akassoglou and macrophage raw264. Also, similar to primary microglia, they were negative for gfap and gc antigens, markers. Including cell culture basics, equipment, protocols and troubleshooting. The present study investigated the antineuroinflammatory and neuroprotective effects of gyejibokryeonghwan gbh, a traditional korean medicine, in lipopolysaccharide lps stimulated murine bv2 microglia. Bv2 cells are a useful cell line because they can be maintained in culture, yet they keep many of the functions and features that microglia express in vivo henn. With the use of the t75 flask i am able to recover about 1015 million cells after 34 days from just 12 million cells originally. Bv2 cells were cultured in dmem gibco, brl, paisley, uk supplemented with 10% fetal calf serum fcs and 100. For noncommercial investigative use only properties. The proper procedure for genome editing on tissuecultured hipscs is described in this protocol. Warm cell culture medium dulbeccos modified eagle medium. Interestingly, grm1 is generally thought not to be present in bv2 cells, yet it was the only one that we significantly detected. Lipopolysaccharide lps stimulated bv2 cells to adopt a microglialike phenotype including an amoebaelike shape, iba1 positive staining and il1. Protocol for hela cell culture and plasmid transfection av02hela cell culture 1. Detection of cell viability and examination of the silencing efficiency of cf in bv2 cells.

We did find low, nonsignificant levels of grm3, 6, and 8. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by 0. Protocol mycoplasma detection test mycoplasma refers to a genus of bacteria that lack a cell wall. Furthermore, bv2 cells were positive for mac1 and mac2, but negative for mac3 antigens. Trem2 inhibits inflammatory responses in mouse microglia. Use of life technologies dmem in the culture of bv2 cells. Primary cultures of hypothalamic neurons treated with culture medium from lps treated bv2 cells increased crf, crfr, pcreb and camp levels compared to. Crisprcas9directed genome editing of cultured cells. Microglia, the central nervous systems innate immune cells, mediate neuroinflammation and are implicated in a variety of neuropathologies. In vitro examination of microglianeuron crosstalk with bv2 cells. Cell culture primary mouse microglia, mouse astrocyte, neuro2a mouse neuroblastoma, 293t, nih3t3 and hela cells were cultured in dmem cellgro supplemented with 10% fetal bovine serum fbs and penicillinstreptomycin invitrogen. Bv2 cells appear have a diverse receptor and signaling gene product profile.

In brief, 3 hours after culture, media was replaced, followed by subsequent media changes and 1. Culture of the cells was based on a modified protocol 30. The suitability of bv2 cells as alternative model system for. Continue to maintain the cells in culture until the viability of the recovered cells is confirmed see step 9. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. Cell culture protocols thermo fisher scientific kr. Protocol for immunofluorescence staining of adhesion cells. Cell culture basics handbook thermo fisher scientific. Briefly, bv2 cells were cultured in dmemhg media containing 10% fetal bovine serum, 100 iuml penicillin, and 100 mgml streptomycin.

The use of t75 flasks in culture of bv2 cells biocompare. Fundamental techniques in cell culture sigmaaldrich. Protocol for immunofluorescence staining of adhesion cells this is provided as a general protocol. Cell culturing of human and murine microglia cell lines will be described in this chapter. By in vitro culture of n9 and bv2 cell lines and lipopolysaccharide lps stimulation model, generation and release of prongf, probdnf and mmp9 was studied in the activated microglial cells by immunocytochemistry, western blotting and bioassay methods. The cells were maintained in high glucose dulbeccos modified eagles medium dmem supplemented with 10 % fetal bovine serum, 100 unitsml penicillin, and 100 mgml streptomycin.

The percentages of apoptotic cells were determined by flow cytometry. Keep cardboard packaging to a minimum in all cell culture areas. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Cell viability was assessed via mtt assay according to the manufactures protocol. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Protocol for hela cell culture and plasmid transfection av. Microglia as a surrogate biosensor to determine nanoparticle. Visit our website for a detailed protocol on using a hemocytometer to obtain a viable cell count 6. Research open access reciprocal interactions of mouse bone. Test cells for the presence of mycoplasma on a regular basis. Remove medium from one dish flask, wash and trypsinize as written in the cell culture guidelines.

A rapid and sensitive method for the quantitation of microgram. Activation of microglial cells was observed with obvious increasing iba1immunoreactivity following lps stimulation in cell culture. Most cell lines will grow on culture flasks without the need for special matrixes etc. Heidcamp, gustavus adolphus college a large collection of protocols related to cell biology work from microscopy to cell culture, histochemistry, etc. 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. Henn a1, lund s, hedtjarn m, schrattenholz a, porzgen p, leist m. Optimize your cell culture technique with proteintechs complete guide. Obtain frozen stock of bv2 microglial cells at passage 18 25 from storage at 80 c. Permeabilization if the target protein is intracellular, it is very important to permeabilize the cells. The differences may be due to differences in the activation state of the cell lines.

Protocol for freezing cells when you have started a new cell line it is a good policy to freeze down a good portion of the cells for use at a later date. I have been trying to maintain this cell line for the past 1 year but they just keep dying. Grow cells on glass coverslips or prepare cytospin or smear preparation. Tetrandrine suppresses lipopolysaccharideinduced microglial activation by inhibiting nfkb and erk signaling pathways in bv2 cells yalong dang1,2,3, yongsheng xu2,3, wentao wu2, weiyi li2,3, yanran sun3, jing yang1, yu zhu1. Bv2 cells were cultured as described previously dai et al. In particular, we will focus our attention on nerve cell cocultures and their uses in studying neuroinflammatory process. We selected the 120 split from a few days previous. Ideally, this cell culture protocol will result in astrocytes reaching confluency on days 910. Count the cells using trypan blue for a viable cell count. Atcc offers custom testing services for mycoplasma detection and human cell line authentication str analysis.

Microglia are both a source and target of extracellular cyclophilin a. Autophagy down regulates proinflammatory mediators in bv2. Protocol for primary microglial culture preparation ncbi nih. Culture of hek 293293t cells charles yin last updated. Useful numbers for cell culture thermo fisher scientific. Primary murine microglia were isolated and cultured from c57bl6 j mice using a previously reported protocol with slight modifications. Antiinflammatory effects by pharmacological inhibition or.

Cell viability was determined by cellcounting kit8 cck8. For example, if you thaw a vial of cos cells to carry the cell line you will eventually split the cells into 1015 plates. Inhibition of inducible no synthase, cyclooxygenase2 and. Tetrandrine suppresses lipopolysaccharideinduced microglial. Centrifuge conical tube containing cells at 1100 rpm for 2 minutes at room temperature. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Cells are then repassaged into a new flask containing the same media every 34 days. Dihydroartemisinin inhibits multiplication of brucella suis. Cell culture guidelines the following is a general guideline for culturing of cell lines. Here you can find useful information about the bv2 cell line. An overview of in vitro methods to study microglia. The suitability of bv2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation.