Oligodendrocyte Progenitor Cell Starter Kit
Contents
1. in OPC K 1 x 40 um 1 x 40 um 1 x 40 um MDS 1 x 8 ml in OPC K 5 10 min 37 C 2 ml 60 mm PL dish 5 10 min 37 C 2 ml 60 mm PL dish 5 10 min 37 C 2 ml 60 mm PL dish BSM 1x30 ml 1x30 ml 1x 30ml OGM in OPC K amp OPC SK 1x30 ml 1x30 ml 1x30 ml OSDM for mature OLs 30 ml not included 30 ml not included 30 ml not included Perform these steps if you want to e increase OPC number in growth medium OGM or OGM pro on PL dishes e eliminate possible microglia contamination on MP dish e obtain mature OLs in differentiation medium OSDM Without cell strainer Alternatively let undissociated tissue pieces settle down by gravity for 10 min at 4 C Carefully transfer the single cell suspension into a new tube and centrifuge Benefits at a glance e cost effective and time saving at least fourfold e animal saving gt 50 e easy handling no longer than 2 hours work on the bench at Day 0 e no dedicated equipment required e high cell yields from postnatal brains up to 1 x 10 cells within 4 days e the unique MAD properties differential brain cell adhesion and target cell isolation e gt 90 pure OPC population within 2 days e high OPC number due to the unique properties of OGM OGM exp and OGM pro oligodendrocyte growth media e oligodendrocyte survival and maturation due to the unique properties of OSDM Please no2. adherent in the dish Transfer cell suspension into a 15 ml tube and carefully add dropwise 6 ml Basal Support Medium Agitate the dish and collect residual progenitor cells Collect cells by centrifugation 5 min 390 x g and remove the entire supernatant to avoid dilution of CDS 3 solution with residual medium in the tube Since isolated cells tend to aggregate resuspend cell pellet in 100 ul CDS 3 and leave for 10 15 min at RT Disaggregate cells by slowly pipetting 6 times up and down with a 200 ul micropipette to obtain single cell suspension and then dilute in culture medium Resuspend cells in 5 6 ml Oligodendrocyte Growth Medium OGM OGM exp or OGM pro and plate cell suspensions into your final dish or slide format poly L lysine coated dependent on your specific application For optimal cell adhesion and OPC proliferation we strongly recommend plating MAD derived cells onto a poly L lysine coated surface Please note that OPCs might be poorly adhesive on poly D lysine coated surfaces E OPC expansion and further applications Dependent on your specific application s OPCs collected by centrifugation can be further cultured on poly L lysine coated coverslips or culture dishes in defined serum free media provided by P Glia Change culture medium every 2 days a in OGM if you wish to expand NG2 glia OPCs b in OGM exp if you wish a rapid expasion of NG2 glia OPCs c in OGM pro if you wish to keep the proliferative cell
3. HBSS 10 10 11 12 13 14 15 16 Separate forebrains from the rest brain regions and transfer into a HBSS containing culture dish 2 forebrains dish No need to remove meninges All the following steps are performed ina sterile laminar flow Aspirate HBSS and add 1 ml CDS 1 at room temperature RT Quickly disaggregate the tissue with fine tweezers then gently pipette tissue pieces 2 3 times in the enzyme solution with an unpolished sterile pasteur pipette to obtain pieces of about 2 3 mm and transfer tissue pieces with the enzyme solution into a 15 ml tube using a sterile pasteur pipette Incubate for 8 min at RT and gently rock the tube from time to time to provide an even enzyme access to the tissue Immediately add 6 ml ice cold HBSS close the tube and mix the content Collect cells and tissue pieces by centrifugation 5 min 390 x g 4 C If not done at the beginning see 1 prepare a fire polished pasteur pipette with an aperture of 0 5 1 mm in diameter using a gasburner and keep the pipette in a sterile tube until the end of the cell isolation procedure optional Aspirate supernatant very carefully the tissue pellet is usually not very compact using the unpolished pasteur pipette and add 1 ml CDS 2 Incubate for 5 min at RT and rock the tube from time to time Dissociate tissue pieces with the fire polished pasteur pipette by gently pipetting up and down 6 times only to obtain single cell
4. from brain cell suspensions see also under your specific applications on page 12 See also P Glia website for related products and applications Introduction In addition to neurons the brain contains three main types of glial cells astrocytes oligodendrocytes and microglia Glial cells significantly contribute to the health and disease state of the central nervous system CNS and a number of neurodegenerative disorders have been associated with glial cell dysfunction 1 The application of glial cell culture techniques for the analysis of their cellular and molecular properties has therefore become a promising tool in biological and medical sciences 2 Particularly for oligodendrocytes rapid and reliable methods for the isolation and culture of high yield pure and viable cell populations at distinct differentiation stages are yet missing Oligodendrocytes are the myelin forming cells in the CNS whose main function is the insulation of axonal processes by the formation of the myelin sheath During late embryonic and early postnatal development of the mammalian forebrain oligodendrocytes arise in the subventricular zones of the lateral ventricles from oligodendrocyte progenitor cells OPCs OPCs migrate out from these germinal zones to populate the developing white and gray matter There cells become postmitotic and differentiate into mature oligodendrocytes of multipolar morphology 3 4 5 An overview on the molecular marker exp
5. target cells to very strong After 2 days in culture of brain cells in growth medium OGM weak cellular interactions with the MAD surface can be disrupted by agitation of the MAD O dish and cells collected by centrifugation How it works For NG2 glia OPC isolation all you need to do is to dissociate early postnatal brain tissue and plate single cell suspensions into MAD O in a specialized oligodendrocyte growth medium OGM or OGM pro Then just let brain cells undisturbed for two days in culture The concept of the MAD O surface is such that OPC interactions and adhesion are the weakest ones Upon short agitation of the culture dish OPCs can easily be detached from MAD and collected by centrifugation The procedure allows an easy and reliable isolation of NG2 glia oligodendrocyte progenitors It has been optimized for early postnatal mouse or rat brain tissue The flow charts below Figure 3 give a short cut of the bench protocol and further culturing options for MAD O derived NG2 glia progenitor cells Day 0 dissociate cells from P3 forebrains 4 Day 0 remove myelin by density centrifugation Day 0 plate cells into MAD O in growth medium 4 Day 2 harvest NG2 glia OPCs NG2 A2B5 O4 Culture in OGM Culture in OSDM growth medium differentiation medium A OPC mature OL NG2 A2B5 04 GalC MOG GalC MBP PLP Figure 3 Bench protocol upper panel and further OPC applications lower panel
6. MAD derived OPCs can be further expanded in OGM or OGM exp oligodendrocyte growth media on PL dishes or directly differentiated into mature OLs for further 3 6 days in OSDM oligodendrocyte survival amp differentiation medium For further details see OPC K Manual Purity and cell yields Since the control of OPC detachment from MAD under a microscope by agitation can be very individual dependent the number and purity of isolated cells after two days may vary What you get The following Figure 4 shows MAD O derived cells you get NG2 glia oligodendrocyte progenitors after culture in OGM growth medium OPCs from P3 mouse brain expanded for 2 days in OGM on a PL dish Figure 4 MAD for oligodendrocyte progenitor cell isolation P3 mouse forebrain derived cells were grown for 2 days on MAD O in Oligodendrocyte Growth Medium OGM and OPCs were collected from the dish by agitation MAD derived OPCs were plated into PL poly lysine containing dishes and expanded in OGM for further 2 days Three specialized oligodendrocyte media help to meet your particular needs e Oligodendrocyte Growth Media OGM OGM exp OGM pro are specialized serum free media optimally supplemented with growth factors to support rapid OPC NG2 A2B5 04 expansion e Oligodendrocyte Survival amp Differentiation Medium OSDM is a unique serum free medium containing neuronal factors and supporting both OL survival and differentiation into m
7. Support Medium 1ml tube with a micropipette and collect cell suspensions into one tube Collect cells by centrifugation 5 min 390 x g 4 C 11 D OPC propagation and isolation 1 Day 2 Resuspend cell pellet in 1 ml ice cold Oligodendrocyte Growth Medium OGM or OGM pro and add the cell suspension to the MAD O dish containing 6 ml Oligodendrocyte Growth Medium Mix the dish content to allow an even distribution of brain cells over the substrate surface and incubate MAD O for 1 hour in a humidified 37 C 5 CO incubator Carefully remove the medium containing floating cellular material and wash MAD O once very carefully dropwise with 6 ml Basal Support Medium Remove the entire medium Apply dropwise 6 5 ml fresh Oligodendrocyte Growth Medium to the dish and leave cells undisturbed for 2 days in vitro 2 div in a humidified 37 C 5 CO incubator Examine cell culture under a microscope and agitate the dish A lot of proliferating oligodendrocyte progenitors progenitor cell aggregates either adherent or floating in the medium less other brain cells astrocytes microglia meningeal cells and some myelin debri brain age dependent are present in the dish Agitate the dish in the culture medium for lt 1 min by controlling cell detachment from MAD under a microscope Do not pipette Most of the oligodendrocyte progenitors progenitor cell aggregates readily detach from the MAD O surface while other brain cells should remain
8. ature MOG GalC MBP PLP myelin forming cells The isolation procedure has been optimized for postnatal day 3 P3 mouse brain tissue and also applies to P2 rat and P7 8 mouse brains respectively In order to obtain higher cell yields and reduce myelin contamination it is strongly recommended to exactly stick to the parameters i e enzymatic treatment density centrifugation parameters number of culture dishes etc worked out for the number and age of animals given in Table 2 on page 9 With P Glia media and reagents you have of course no limits for your scientific creativity towards optimizing your personal needs of animals brain age and region etc Table 2 Number of postnatal animals and amounts of reagents required Treatment Reagent 2 x P3 mouse forebrains 2 x P2 rat forebrains 1 x P7 mouse forebrain CDS 1 1x1ml 8 min RT 1x1 ml 8 min RT 1x1 ml 15 min RT CDS 2 1x1ml 5 min RT 1x1ml 5 min RT 1x1ml 5 min RT CDS 3 1x 100 ul 10 15 min RT 1x 100 ul 10 15 min RT 1x 100 ul 10 15 min RT SDM centrifugation 2 x 6 2 ml cushion 2 x 6 2 ml cushion 2 x 6 5 ml cushion MAD O culture 1x 95 mm dish 2 days 1 x 95 mm dish 2 days 1x 95 mm dish 2 days PL dish culture in OPC K 1 x 60 mm dish 2 days 2 x 60 mm dish 2 days 1 x 60 mm dish 2 days MP dish not included 1 x 95 mm dish 30 min 1 x 95 mm dish 30 min 1 x 95 mm dish 30 min Cell strainer
9. ctivity If thawed during shipment and not going to be used within one week freeze again at 20 C in working aliquots until use Always thaw overnight at 2 8 C Avoid repeated freezing and thawing which will impair the activity of culture media Before storage at 2 8 C incubate at 37 C to redissolve all ingredients and obtain a clear solution Always shake the bottle before use Table 2 on page 9 Additional P Glia media amp reagents needed would depend on your specific applications and can be purchased separately see page 4 and page 12 Note N be FP Additional Materials Required e HBSS Hanks balanced salt solution without calcium chloride and magnesium sulfate sterile filtered e PBS pH 7 4 10 mM phosphate buffer pH 7 4 140 mM NaCl 3 mM KCl sterile filtered e sterile instruments for tissue preparation fine scissors tweezers forceps e sterile tubes 15 and 50 ml and culture dishes 60 mm e sterile serological pipettes 200 ul and 1 ml micropipettes and pipette tips e sterile pasteur fire polished pasteur pipettes optional e cell strainer 40 um optional e standard cell culture equipment inverse microscope centrifuge optimally with a swing out rotor laminar flow humidified 37 C 5 CO incubator e Attention Adjust speed 390 1 x g and time 5 min of your lab centrifuge and keep these parameters throughout the whole isolation procedure Additional P Glia media and reage
10. e O Glia Neural Cell Solutions in CNS Research Oligodendrocyte Progenitor Cell Starter Kit User Manual Cat No OPC SK Life could be that easy NG2 glia OPCs within two days Check all kit components immediately upon arrival for completeness and storage conditions FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC AND THERAPEUTIC USE May 2013 P Glia P Glia Website www pglia com Email info pglia com Table of Contents Kit Components and Storage Additional Materials Required Introduction Principle and Benefits Protocol A General considerations mone Abbreviations BSM CDS 1 CDS 2 CDS 3 CNS GalC MAD O MBP MDS MOG OGM OSDM OL OPC OPC exp PDGF AR PLP RT SDM Tissue dissociation Isolation of single cell suspensions OPC propagation and isolation OPC expansion and further applications basal support medium cell dissociation solution 1 cell dissociation solution 2 cell dissociation solution 3 central nervous system galactocerebroside multiple adhesion dish for oligodendrocytes myelin basic protein mild dissociation solution myelin oligodendrocyte glycoprotein oligodendrocyte growth medium oligodendrocyte survival amp differentiation medium oligodendrocyte oligodendrocyte progenitor cell OPC expansion dish platelet derived growth factor alpha receptor proteolipid protein room temperature separation density medium Dn UH A W 10 10 11 12 12 Kit C
11. hange of any parameters is not recommended and no responsibility will be taken accordingly To facilitate the step by step performance avoiding unprepared handling some preparatory steps in italic bold have also been included The whole procedure takes about 1 5 2 hours from tissue preparation to culturing of brain cells Attention All P Glia dishes should be washed 2 x 10 min with PBS before use Before starting with cell isolation place MAD s at RT and rehydrate for 10 min in PBS Wash once with PBS and keep in Oligodendrocyte Growth Medium 6 ml 95 mm dish in a humidified CO incubator until use B Tissue dissociation Day 0 1 Prepare one unpolished and one fire polished sterile pasteur pipettes with an aperture of 0 5 1 mm in diameter using a gasburner and keep the pipettes in 15 ml sterile tubes until the end of the cell isolation procedure Prepare one 50 ml sterile tube for waste 2 Decapitate postnatal mice rats and collect heads in a sterile dish Decontaminate the entire surface of the skin with 70 alcohol and wait for 3 5 min to ensure it is sterile 3 Inthe meantime prepare 2 x culture dishes 60 mm with HBSS 2 ml dish 4 Carefully remove the skin from the cranial region of each head 5 Cut out the dorsal part of each skull very carefully with sterile scissors Avoid disruption of the underlying brain tissue 6 Dissect out brains with sterile tweezers and collect them into a culture dish containing 2 ml
12. iple of neural cell isolation relies on basic knowledge in cell biology that the strength of cell adhesion to the surrounding matrix or to other cells strongly depends on the affinity of different cellular receptors to their extracellular ligands and the respective activation of intracellular signaling Ligand receptor interactions may thus result into diverse adhesive forces varying from very stable to weak or even repellent The unique properties of MAD O are based on accordingly selected multiple cell binding sites supporting the adhesion of different cell types and of different adhesive strength Taking an advantage of such molecular mechanisms the MAD O surface allows the isolation NG2 glia OPCs out of a complex brain cell mixture due to the weak adhesiveness of target cells Moreover this takes place under defined culture conditions favoring the growth of the desired cell type Figure 2 Brain cells ss target cell OPC 2 days in culture in OGM Cellular interactions O T B o weak stronger strongest maoo ii i t e itt contains multiple cell binding sites t t ft MAD agitation OPC oligodendrocyte progenitor cell OPCs of OGM oligodendrocyte growth medium MAD O multiple adhesion dish for OPCs Figure 2 MAD principle or the power of being weak The unique properties of MAD are based on multiple cell binding sites supporting the adhesion of different cell types and of different adhesive strength from weak of the
13. nts which can be purchased separately if needed Cat No Description Size Storage OGM Oligodendrocyte growth medium is a serum free chemically 50 ml 20 C defined medium specially formulated to support the growth of oligodendrocyte progenitor cells OPCs for 2 3 weeks in vitro OGM exp Oligodendrocyte growth medium exp is a serum free 50 ml 20 C chemically defined medium specially formulated to support the rapid expansion of OPCs in culture P Neural P Glia defined neural medium is a chemically defined medium 50 ml 20 C formulated for culturing of different neuronal and glial cells cell lines under serum free conditions OSDM Oligodendrocyte survival and differentiation medium is a 30 ml 20 C unique serum free medium specially formulated to support the survival and terminal differentiation of brain derived OPCs into mature oligodendrocytes OLs MAD O Multiple adhesion dish for oligodendrocytes contains 4x95mm 20 C multiple cell binding sites specially formulated for OPC isolation from mouse or rat brain cell suspensions after 2 days of culture in OGM PL D Poly lysine containing dish allows the rapid expansion of 8 x 60mm 2 8 C MAD derived OPCs within 2 days of culture in growth medium PL containing dishes can also be used for culturing of other primary neural cells MP D Microglia preadhesion dish contains cell binding sites allowing 4x95 mm 20 C the rapid adhesion and removal of contaminating microglia
14. omponents and Storage Please examine all kit components immediately upon arrival for leakage or breakage Notify P Glia or your distributor immediately if any problems occur Please note that if kit components thaw during shipment their activity remains unaffected if they are stored at the adequate temperature see Table 1 notes 1 2 Except for Separation Density Medium which should be stored at 2 8 C all other kit components must be transferred immediately to a freezer and stored at 20 C until use Once thawed cell culture media must be stored at 2 8 C as indicated in Table 1 Do not refreeze Table 1 OPC Starter Kit components and storage conditions Cat No CDS 1 CDS 2 CDS 3 SDM BSM OGM MAD O Manual OPC SK is a basic starter kit for OPC isolation from early postnatal mouse or rat brain for overview see Description Amount Cell dissociation solution 1 1ximl Cell dissociation solution 2 1x1ml Cell dissociation solution 3 1x 100 ul Separation density medium 1x20 ml Basal support medium 1x30ml Oligodendrocyte growth medium 1x20 ml Multiple adhesion dish OL 1x95 mm OPC Isolation Kit 1x manual Storage Stability 20 C for 12 months 20 C for 12 months 20 C for 12 months 2 8 C for 12 months 20 C for 12 months 2 8 C for 1 month 20 C for 12 months 2 8 C for 2 weeks 20 C for 6 months 1 x check list If thawed during shipment freeze again at 20 C until use No loss of a
15. ression along the oligodendrocyte cell lineage is given in Figure 1 es early O 2A OPC late OPC pro OL immature 04 OL mature OL GD3 A2B5 NG2 GD3 A2B5 04 R3int A2B5 04 GalC MBP MOG GalC MBP PLP Biint t PDGF AR Figure 1 Molecular phenotype of oligodendrocyte OL lineage cells Early O 2A OPCs express gangliosides GD3 A2B5 NG2 chondroitin sulfate proteoglycan 81 integrins B1 int and PDGF alpha receptor subunit PDGF AR Late OPCs pro OLs express gangliosides 83 integrin B3 int and O4 antigen sulfatides and have lost NG2 and 1 integrin expression Further lineage progression of immature 04 OLs is characterized by the sequential expression of myelin specific glycolipids sulfatides and GalC and proteins MOG MBP PLP involved in their terminal differentiation into mature myelin forming cells References 1 Schmitz T Chew LJ 2008 Cytokines and myelination in the central nervous system ScientificWorld Journal 8 1119 1147 2 van Noort JM 2006 Human glial cell culture models of inflammation in the central nervous system Drug Discovery Today 11 74 80 3 Miller RH 1996 Oligodendrocyte origins Trends Neurosci 19 92 96 4 Nave KA 2010 Oligodendrocytes and the micro brake of progenitor cell proliferation Neuron 65 577 579 5 Richardson WD Kessaris N Pringle N 2006 Oligodendrocyte wars Nat Rev Neurosci 7 11 18 Principle and Benefits The MAD multiple adhesion dish princ
16. state for several weeks d in P Neural if you wish to retain an immature phenotype for at least 3 days e in OSDM if you wish to obtain mature OLs expressing myelin proteins MOG MBP PLP and glycolipids GalC within 3 6 days For further details and treatments see also OPC K Manual pages 12 13 12
17. suspensions f no gasburner is available to prepare a fire polished pasteur pipette use a 1ml micropipette instead Avoid foaming of cell suspensions which may disrupt living cells Don t worry if cell aggregates are still present these are mostly meningeal tissue pieces Add 6 ml ice cold Basal Support Medium and mix the tube content If no 40 um cell strainer available let undissociated tissue pieces settle down by gravity for 10 min at 4 C carefully collect the supernatant with the fire polished pasteur pipette into a new 15 ml tube and centrifuge In the meantime prepare 2 x 15 ml tubes with ice cold Separation Density Medium 6 2 ml tube see Table 2 for density centrifugation Shake the SDM bottle before use C Isolation of single cell suspensions 1 Aspirate supernatant and resuspend cell pellet in 1 ml ice cold Basal Support Medium Adjust the volume of the single cell suspension to 4 ml with Basal Support Medium Apply 2 ml cell suspension with the fire polished pasteur pipette on top of each tube with Separation Density Medium very slowly to avoid intermixing with the underlying solution and centrifuge 5 min 390 x g 4 C Get sure that MAD O was rehydrated and placed into a humidified CO incubator see instructions on p 10 Carefully aspirate the supernatant from each tube by first aspirating the upper 5 ml containing cellular and myelin debri and then the rest medium Resuspend cell pellets in ice cold Basal
18. te that in primary cell cultures the final cell yields may vary dependent on animal species number and age of animals used between individual animals operator s skills and or additional materials used Protocol PLEASE READ THIS PROTOCOL CAREFULLY BEFORE STARTING WITH CELL ISOLATION A General considerations You should be aware of the following safety rules concerning aseptic handling of primary animal tissue and cell culture materials 1 Decontaminate all surfaces of workbenches microscope s laminar flow etc going to be used for tissue preparation and cell culture with 70 alcohol to ensure they are sterile Decontaminate external surfaces of all vials bottles and micropipettes with 70 alcohol to ensure they are sterile Make sure all solutions cell culture materials and instruments for tissue preparation are sterile Always thaw cell culture media overnight at 2 8 C see Table 1 for further details Perform cell culture work only in a sterile hood or laminar flow Do not pipette with mouth Always wear protective gloves and lab coat when working with primary animal tissue After thawing always keep enzyme solutions on ice until use m OD e The following protocol is recommended for OPC isolation using 2 forebrains from P3 mice or P2 rats Amounts of solutions and incubation times indicated at different steps of this protocol have been optimized exactly for the number of postnatal animals used see Table 2 page 9 C
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