Themes/Projects

--- title: Themes/Projects disqus: hackmd --- Themes/Projects === :::success > Documentation [name=MrDr.Staffan] ###### tags: `page`, `Astrocyte_culture` ::: :::info ### Table of Contents [TOC] ::: [Top](#Table-of-Contents) **Why this page?** Astrocyte culture --- # siRNA Ask John: All the targetd sequences are the same. It costs £200 more to get them in different tubes. Do we get the actual sequence of the contstruct? Delivery Fuse-It-siRNA infrared flourescent ready to use, 6mM 2 x 300 ul https://www.thistlescientific.co.uk/product/fuse-it-sirna/ miRNA Bbox transcript 1+2 3+4 miRNA control gapdh Custom miRNA https://horizondiscovery.com/en/gene-modulation/knockdown/sirna/products/on-targetplus-sirna-reagents ![](https://i.imgur.com/O0V6Ilf.png) SMARTpool: A mixture of 4 siRNA provided as a single reagent; providing advantages in both potency and specificity. Set of 4: A convenient option for purchasing aliquots of all 4 individual siRNAs targeting a single gene. Individual siRNAs: Select 1, 2, or 3 individual siRNAs per gene. ## CreSi They also do custom RNAsynthesis. Why not try my idea of Endogenous silencing? CreSi EnSAM Endogenous Silencing by Anti-microRNA ` "inluding a stretch of sequence that is a target for an endogenous miR" in my case. a) If I want expression only in brain, then the mRNA for my transgene include a silencing sequence for a mRNA that is expressed in the tissue where my promtoer is also active. If I want to silence only in the brain, then the silencing vector includes a target sequence for being silenced in other cells.` # **Detection** Ketone assay https://www.sigmaaldrich.com/catalog/product/sigma/mak134?lang=en&region=GB MAK134 Free Fatty Acid Quantitation Kit MAK044 # **Compounds** C2 Sodium acetate S5636-250G **Ordered** C3 Sodium propionate - propionic acid P5436 **Ordered** C4 Sodium butyrate B5887-250G **Ordered** C5 Pentanoate - Valeric acid C4026-10G C5 Valproate branched=2xC5 Valproic acid P4543-10G **Ordered** C6 hexanoate C4026-10G C7 heptanoate only find it with short chain alcohols e.g. ethylp-heptanoate C8 Sodium caproate - Octanoic acid C5038 C10 Decanoate C4151 # Emails :::success Email: 21 Oct 2020, 17:19 Amv Hi Staffan, So I did 2 brain for astrocyte isolation and get **1,05 Million per brain** which is quite ok. I use for Ab what was described in the protocol: 80µl buffer + 20µl Ab-GLAST-biotin then 80µl buffer + 20µl Ab-Biotin-magnetik. It seems to work well like this. I plate 3 plates: a full 24-well plate, a full 6-well plate and a half 6-well plate. Let see tomorrow or Friday if they look nice ! ::: 20 Nov 2020, 10:53 Staffan John: I will look out for those genes going through my material. If I remember correctly there are direct links to iron homeostasis and bilirubin synthesis. Will try to lure those back into mind. I also noticed B27 actually contains Carnitine... Adrien: Let's get the compounds in first. Whenever we have these we can see what will be appropriate. I dont expect increased myelin synthesis per se. But rather ask "if supporting OPC with appropriate FAs will help them mature?". I might order some "standards kits" of FAs and corresponding Carnitine-conjugates from Sigma e.g. Unsaturated https://www.sigmaaldrich.com/catalog/product/supelco/un101kt?lang=en&region=GB Odd chain https://www.sigmaaldrich.com/catalog/product/supelco/oc91kt?lang=en&region=GB Saturated https://www.sigmaaldrich.com/catalog/product/supelco/ec10a1kt?lang=en&region=GB I am reading these to get an introduction to myelin synthesis, perhaps a zoom talk later. https://pubmed.ncbi.nlm.nih.gov/29410529/ https://www.lipidmaps.org/resources/lipidweb/index.php?page= Happy for input on considering specific FAs / myelin intermediates. :::success This isn’t a great study as it’s a cell line which is why FBS is used. Not very oligodendroglial like I’m afraid and I would be careful about drawing any conclusions. I forget the common name of docoshexanoic acid but studies have used this. Probably as good as green tea. I think some of these polyunsaturated FA contribute to FA synthesis and also as membrane lubricants during compaction. Not a very clear concise picture yet. However look at the papers David, Hiroko and I previewed for Cell Stem Cell. Very interesting on the cholesterol pathway as it is so energy expensive to synthesise. An old paper involving Claus Armin Nave fed cholesterol to PLP mutant mice and it rescued their phenotype but I think this result may have been exaggerated. But on cholesterol, it’s synthesis is De Novo in the brain, 80% of brain cholesterol is in myelin. Cholesterol, although a lipid behaves as a lubricant as it confers rigidity in membranes when hot and fluidity when cold but also helps curvature. Also a lot of lipid second messengers act on nuclear hormone receptors with estrogen, T3 and retinoic acid. One example from Robin’s lab is RXRg which the drug Bexarotene is being repurposed for. Patrizia Cassacia has also done some work on lipid regulation of histones in OPCs which I think primes them. FAs in B27 include linolenic, linoleic and oleic acid (essential FAs). I think give it a shot but not sure what your readout will be. If its more differentiation then read the papers by Madhaven and Hubler. I think you should work with either myself, Adrien or Guy for a differentiation study. Not saying you can’t do it but I think it looks easier than it is. If however its compaction then you would need very mature Oligos (> 7 d.i.v.), very sparse and stain for MBP (compact), CNP (hemichannels) and have some cell mask plasma membrane for automation of cell segmentation. Ask John, he was trying for me. ::: --- # Lab reference cultures ## 2014 Molofsky Astrocyte-encoded positional cues maintain sensorimotor circuit integrity https://www.nature.com/articles/nature13161 Astrocyte isolation by flow cytometry Postnatal day 7 spinal cords were microdissected using an ‘open book’ preparation to separate dorsal and ventral halves. DRGs and meninges were removed then dissociated with **papain** (20 U ml−1; Worthington) for 80 min at 33 °C as described previously17. Aldh1l1-positive and -negative cells were sorted as previously described18 on a BD Facs Aria II and gated on forward/side scatter, live/dead by DAPI exclusion, and GFP, using GFP-negative and DAPI-negative controls to set gates for each experiment. In some cases GFP-positive populations were re-sorted using the same gates to >95% purity. Astrocyte cell culture Dorsal and ventral spinal cords from P0–1 mice were isolated and dissociated as above. Cells were plated at a density of >1 × 106 per 25 cm2 flask in **DMEM-hi glucose with 10% FCS/10 μM hydrocortisone, 5 μg ml−1 N-acetylcysteine, 2 μg ml−1 insulin and 20 ng ml−1 EGF**. Six days after plating cells, flasks were shaken to remove oligodendrocyte contamination. At 8 days, AraC was added to kill rapidly proliferating cells. For DRG co-cultures, 10–12 days after initial plating cells were re-plated into assay containers that consisted of 8-well glass chamber slides (BD) coated with poly-D-lysine and recombinant human fibronectin (Biomedical Technologies) to promote astrocyte adhesion, plating 30,000 cells per well. In most cases, cultures were established using Sema3afl/fl mice, and adenoviral Cre recombinase (Vector Biolabs) was added to some wells 2–4 h after re-plating. For MN co-cultures, astrocytes were re-plated at 2,000 cells per well onto a reduced GF Matrigel substrate (BD) diluted 1:25 in DMEM. Astrocyte monolayers were then cultured for 2–3 days before adding neurons. DRG isolation and co-culture DRGs from E13.5–14.5 mouse embryos were isolated and dissociated for 45 min in 0.25% trypsin/EDTA (Invitrogen). Five-hundred cells per well were plated onto astrocyte monolayers in minimal neural growth media containing DMEM:F12, 10% FCS, N2 and B27 supplements (Invitrogen), and co-cultured for 48 h before fixation in 4% paraformaldehyde and immunolabelling. Motor neuron isolation and co-culture Spinal cord neurons were isolated from embryonic rat spinal cords on the basis of previous protocols32. In brief, spinal cords were dissected from E15 rat embryos, dissociated in 0.25% trypsin (Gibco) for 15 min and triturated to form a single-cell suspension in L-15 plus 10% FBS media (Gibco). The suspension was immunopanned in a series of negative selection plates against rat neural antigen 2 (Ran2) and galactocerebroside, and then motor neurons were positively selected for on a final p75NTR panning plate. Adherent cells were released from the plate with a brief application of 0.05% trypsin (Gibco) and re-suspended in growth media (DMEM, B27, N2, Pen-Strep (Gibco)) before culturing. At re-plating (onto Matrigel-plated sub-confluent astrocytes for co-cultures, or Matrigel-coated wells for recombinant Sema3a experiments), fresh media was added consisting of DMEM hi-glucose supplemented with N2 and B27 supplements 5 μg ml−1 N-acetylcysteine, 5 μg ml−1 insulin and 5 μM forskolin. # Random xeno-free A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype https://sci-hub.se/10.1016/j.jneumeth.2019.03.013 2.2. Culture media Astrocytes cultures were prepared in differing media conditions, defined as follows. The serum containing media known as MD medium is composed of Dulbecco’s Modified Eagle’s medium (DMEM with 5.5 mM glucose, 4 mM L-glutamine, 1 mM sodium pyruvate) containing 10% FBS and streptomycin (100 units/ml) - penicillin (100 μg/ml). Serum free media defined as astrocyte based media (ABM) is composed of Neurobasal medium and DMEM (1:1 v/v) supplemented with 5.5 mM glucose, Penicillin (100 units/ml)-streptomycin (100 μg/ml), 1 mM sodium pyruvate, Glutmax, bovine serum albumin (100 μg/ml), transferrin (100 μg/ml), putrescine dihydrochloride (16 μg/ml), progesterone (60 ng/ml), sodium selenite (40 ng/ml), and N-Acetyl Cysteine (5 mg/ml). ABM was supplemented with 2, 5 or 10 ng/ml of Human FGF-basic, Human EGF, or a combination of both. https://experts.unthsc.edu/en/persons/shaohua-yang/publications/ ![](https://i.imgur.com/pdE3ZY3.png) ![](https://i.imgur.com/asFqUrF.png) ## Gene profile FBS vs serum free It was reported that astrocytes cultured in serum free media had a gene profile more representative of in vivo astrocytes as compared with those cultured in FBS containing media (Cahoy et al., 2008; Doyle et al., 2008; Foo et al., 2011). ## Other efforts Foo & Zhang Efforts have been invested in the development of serum-free primary astrocyte culture systems, including a recent method combining negative immunopanning and a serum-free heparin-binding EGF-containing medium which has been demonstrated to resemble astrocytes in vivo (Foo, 2013; Foo et al., 2011; Zhang et al., 2016). # Media https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-formulations.html Cell culture medium formulation and its implications in cancer metabolism https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557711/ ## Factors Aside from molecular component known to perform certain functions attributed to FBS in media (Fischer et al., 1982; Obayashi et al., 2009) transferrin, selenium, hormones, But also Heparin–binding epidermal growth factor (HBEGFF) has been shown to promote astrocyte survival and proliferation and to affect cell differentiation and morphology in serum free culture conditions (Mayer et al., 2009; Puschmann et al., 2014). FGF2, first purified in bovine brain (Gospodarowicz et al., 1982), has been found to maintain astrocyte in nonreactive state Similarly, FGF signaling delays astrogliosis and accelerates astrocytes deactivation after injury (Hizay et al., 2016; Kang et al., 2014; Menon and Landerholm, 1994). ## Osmos? ## Carnitine? Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro https://sci-hub.se/10.1071/RD11262 ## Fattyacid composition? ## B27 https://hannalabweb.weizmann.ac.il/?page_id=129 https://hannalabweb.weizmann.ac.il/wp-content/uploads/2016/02/HANNA-LAB-B22-B27-PROTOCOL-V3.pdf L-carnitine - 80mg / 800ml Oleic acid Pipecolic acid # Goldman: How to make an oligodendrocyte https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712837/ ![](https://i.imgur.com/3ClHeQ2.png) ![](https://i.imgur.com/ezYWrfl.png) ![](https://i.imgur.com/JJnf8nO.png) NT3, neurotrophin 3 (NTF3); OL, oligodendrocyte; RA, retinoic acid; SB, SB431542 (a TGFβ antagonist); T3, thyroid hormone # Khalil's protocol ![](https://i.imgur.com/vTd3nJ8.png) a ![](https://i.imgur.com/0AxSHuV.png) a ![](https://i.imgur.com/bqe8RPX.png) SATO (should already include Apo-Transferrin) 1 100 x 1 x Pyruvate 1 100 mM 1.5 mM Apo-Transferrin* (should already be in Sato – check if it isn’t) 5 1 mg/ml 50 ug/ml N-acetyl cysteine 0.1 60 mg/ml 60 ug/ml Insulin 0.05 4 mg/ml 10 ug/ml B27 2 50x 1x *Pre-make stock: Dissolve 5 mg per 100 ml medium in 5 ml DMEM/F12. (Add 5 ml for 200 ml medium, dissolve 10 mg / 5 ml); sterile filter Growth Factor: HB-EGF (5 ng/mL) (Peptrotech, 100-47) Change 100% medium the next morning, then 50% every 2 d. GF have to be added at 2x final concentration every 2 d. ## SATO Sodium pyruvate (100 mM; Invitrogen 11360-070) 200 µL 1 mM cautionInsulin stock (0.5 mg/mL) 200 µL 5 µg/mL N-Acetyl-L-cysteine (Sigma-Aldrich A8199) stock (5 mg/mL, prepared in DMEM) 20 µL 5 µg/mL Trace Elements B (1000×; Cellgro 99-175-CI) 20 µL 1× d-Biotin (Sigma-Aldrich B4639) stock (50 µg/mL) We use neumo ## Adrien email HB-EGF (a growth factor for the media) https://www.sigmaaldrich.com/catalog/product/sigma/e4643?lang=en&region=GB Anti-GLAST1 (for Rat astrocyte isolation) https://www.miltenyibiotec.com/GB-en/products/anti-glast-acsa-1-microbead-kit-human-mouse-rat.html#for-5x10-sup-8-sup-total-cells or Anti-ACSA-2 (for mouse astrocyte isolation) https://www.miltenyibiotec.com/GB-en/products/anti-acsa-2-microbead-kit-mouse.html#gref ACSA-2 = ATP1B2 https://www.jbc.org/content/292/21/8874.full.pdf If you know what to do, we can order these products and try the isolation on September 17. We can discuss this soon if you want, Best, Adrien Dr A.M. Vaquie Tue, 1 Sep, 13:32 to sh929 Actually we also need this Ab for Rat astrocytes https://www.miltenyibiotec.com/GB-en/products/anti-biotin-microbeads.html#gref # Barbara Book 2019 Culturing astrocytes ## qPCR 12 well Depends on the levels of the mRNA. ## WB 6 well p153 We performed the coculture in the 12-well plates to analyze gene expression by real-time qPCR, while the coculture on 6-well plates was used to analyze protein expression by western blot. ## In vitro Circadian p148 Rhythmicity cannot be determined with methods that evaluate simply whether the data vary over time (e.g., One-way ANOVA). Instead, rhythmic data typically fulﬁll the requirement of being ﬁt with a periodic (e.g., cosine) function for the duration of the experiment. Therefore, the statistical signiﬁcance of the rhythmic gene expression is classically determined by Cosinor analysis. This method was ﬁrst developed and extensively applied to analysis of biological rhythms by Franz Halberg, at the University of Minnesota, to handle short timeseries and sparse data when prior information is available ## Culturing densities |||| |-|-|-| | Cell type | Purpose | | | Neurons| For morphological measurements | typically plated sparsely (1000 to 20,000 cells/ cm2)| For studies of intracellular signaling or gene expression, 500,000 or even one million neurons per culture may be necessary, depending on the abundance of the mRNA or protein of interest and the sensitivity of the assay employed For many biochemical, molecular biology or early developmental studies, cortical neurons can be cultured for 2 weeks or less. However, mature neurons (cultured for at least 3 weeks) are required for studies of excitotoxicity, synaptic transmission, ion channel function, and gene expression. Some serum-free supplements, such as B27, do not support astrocyte proliferation and thus help to keep neuronal cultures relatively pure It is possible to reduce glial cell contamination by treatment with the antimitotic cytosine arabinoside (AraC) at early time points in the culture. it should be used at its lowest effective does (5 μM) and added after 3–4 days of culture. ## Astrocytes Astrocytes develop from late embryonic stages to early postnatal periods. Indeed during the ﬁrst 3 weeks of postnatal development, the astrocyte population expands six- to eightfold in the rodent brain [33, 34]. As few viable astrocytes can be obtained once their development is complete that is, from late postnatal or adult brain suspensions, they derive mostl yfrom early postnatal mice or rats. It should be kept in mind that the developmental stage of astrocytes inﬂuences their transcriptome/proteome and therefore their functions. [# Lol] **An often-used approach to create more mature astrocytes in culture is addition of the cAMP analog, dibutyryl cAMP (dbcAMP) after a few days in culture. dbcAMP induces astrocytic morphological and functional changes [35, 36] that represent astrocyte differentiation [37].** **These changes include expression of astrocyte-speciﬁc proteins glutamine synthetase (GS) and glutamate transporter-1 (GLT-1), as well as almost doubling the uptake of D-aspartate, used as a nonmetabolizable substitute for glutamate [38]** SLC1A2 // GLT-1 // EAAT2 Alternatively, other methods of direct selection and isolation of astrocytes by immunopanning [39] or antibody-based FACS isolation [40] represent new avenues to further investigate the fundamental properties of mature astrocytes. The most frequently used media are DMEM, MEM or F12, often supplemented with serum, glucose, glutamine or a combination of these We previously reported that **serum starvation** of astrocyteenriched cultures from either E15 or E17 rat fetuses induced a signiﬁcant increase in apoptotic cell death, while barely **increased cell death in postnatal astrocytes** [42–44]. Moreover, we found that survival of serum starved astrocytes obtained from E15 fetuses was increased in **presence of conditional medium from postnatal astrocytes** [43]. This suggested that the resistance of neonatal astrocytes to serum **starvationinduced apoptosis depends, at least in part, on the release of soluble factors that promote their survival** Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture https://doi.org/10.1101/2020.04.28.066043 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152221 # Miltenyi ![](https://i.imgur.com/CSTaFj2.png) |||| |-|-|-| |density | 24 well plate 1.88cm2 | 6 well plate 9.4cm2 | |5.32K/cm2 | 24*10K = 240K | 5.32K * 9.4 => 50K * 6 = 0.3M | |13.3K/cm2 | 24*25K = **600k | 13.3K * 9.4 => **125K** * 6 = **0.75**M | |26.6K/cm2 | 24***50K** = 1.2M | 26.6K * 9.4 => **250K** * 6 = 1.5M | ![](https://i.imgur.com/B0hL9zy.png) ![](https://i.imgur.com/YtKlr3e.png) ## Patents Page14![](https://i.imgur.com/R00a0eP.png) https://patents.google.com/patent/WO2003078610A1/fr?assignee=Miltenyi+Biotec+Gmbh&num=100&oq=assignee:(Miltenyi+Biotec+Gmbh)&page=1 https://patentimages.storage.googleapis.com/2e/de/d4/26bfe877acbfa6/WO2003078610A1.pdf Page21![](https://i.imgur.com/FOHMC2S.png) ![](https://i.imgur.com/WeW2slO.png) ![](https://i.imgur.com/IAwJWMF.png) ![](https://i.imgur.com/hHgaQfH.png) ??? ![](https://i.imgur.com/GVmdAuo.png) Flt IL6 [0095] A further aspect comprises a culture medium supplement composition comprising an EGF component, a tankyrase inhibitor that binds to an adenosine subsite of a tankyrase enzyme, with or without a BMP inhibitor component, which can be used as a supplement for a cell culture base medium. The supplement can also include other components discussed herein such as ascorbic acid, L-glutamine and B27 (or surrogates, such as neurobrew-21). The supplement in an embodiment comprises the active ingredients: BMP inhibitor, EGF, a tankyrase inhibitor that binds to the adenosine subsite of tankyrase enzymes, and optionally one or more of Ascorbic Acid, L-Glutamine and B27. https://patents.google.com/patent/WO2019227198A1/en?q=Neurobrew&num=100&oq=Neurobrew In the adhesion culture, a known differentiation inducer can be used. As a differentiation inducer that can be used when further inducing differentiation of specific hypothalamic neurons (dorsal hypothalamic neurons, ventral hypothalamic neurons, etc.) from hypothalamic progenitor cells, ciliary neurotrophic factor (CNTF), Examples include brain-derived neurotrophic factor (BDNF). The differentiation inducer can be appropriately selected depending on the type of target mature cell. Further, the concentration at the time of addition can be appropriately set according to the substance used, the type of the target cell, and the like. For example, when using CNTF to induce dorsal hypothalamic or ventral hypothalamic neurons, a suitable concentration is usually 1 to 200 ng / ml, preferably 2 to 50 ng / ml. Appropriate concentrations when using BDNF to induce ventral hypothalamic neurons (inner ventral nucleus neurons, A12 dopamine neurons, arcuate nucleus neurons, orexin positive neurons, etc.) are usually 1-1000 ng / ml, Preferably, it is 10-200 ng / ml. https://patents.google.com/patent/JP2013017434A/en?q=gfap+cell+culture&oq=gfap+cell+culture Cells were seeded at 75 × 10 4 cells / cm 2 in a poly-lysine-coated dish and cultured at 37 ° C. in a 5% CO 2 gas phase. The medium was replaced with a fresh medium (10% HM / DMEM plus 20 ng / ml EGF and 20 ng / ml FGF) once every 4 days, and cultured for a total of 8 days. https://patents.google.com/patent/JP2013017434A/en?q=gfap+cell+culture&oq=gfap+cell+culture # Container & Shape areas ![](https://i.imgur.com/8gN7Akr.png) ![](https://i.imgur.com/2QuWC6F.png)