Retinal Ganglion Cell Isolation

140-003-194.02 1. 1. Description 4.

Contents 2. Capacity Components 3. Product format macsde www.miltenyibiotec.com www.miltenyibiotec.com Storage Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi

Protocol Description References Example of a separation using the Retinal Ganglion Cell Cell Ganglion Retinal the using of aseparation Example

2.4 2.3 2.2 2.1 1.4 Applications 1.3 1.2 1.1 Isolation Kit Isolation +49 2204 8306-0, 2204 +49 @

miltenyi.com For For and microglia and retinal ganglion cells ganglion retinal Magnetic separation: Positive selection of CD90.1 selection Positive separation: Magnetic separation Magnetic labeling Magnetic preparation Sample requirements instrument and Reagent information Background Separation MACS® of the Principle

at 2–8 light from Store protected The specified number of separations is valid valid is separations of number specified The Particles Anti-BiotinMACSiBead™ mL 2.5 1.25 0.3 All components are supplied in buffer buffer in supplied are components All vial label. vial MicroBeads conjugated to monoclonal mouse mouse monoclonal to conjugated MicroBeads when using 10 retinas (5 rats, age P6–7) per P6–7) per age (5 rats, 10 retinas using when containing stabilizer and 0.05% sodium azide. 0.05% sodium and stabilizer containing Biotin-conjugated monoclonal antibody. antibody. monoclonal Biotin-conjugated freeze. The expiration date is indicated on the on indicated is date expiration The freeze. IgG2a) mouse sample. anti-mouse/rat CD90.1 antibodies (isotype: (isotype: antibodies CD90.1 anti-mouse/rat 68, 5142968, Fax Fax mL Biotinylated Depletion Antibody: Antibody: Depletion Biotinylated mL 25 separations. separations. 25 +49 2204 85197 2204 +49 mL CD90.1 MicroBeads, MicroBeads, CD90.1 mL Bergisch Gladbach, Germany Bergisch Gladbach, : Depletion of endothelial cells cells of endothelial : Depletion mouse and rat and mouse °C. Do not Do °C. +

rat Kit Isolation Cell Ganglion Retinal Antibody and Anti-Biotin MACSiBead™ Particles. The depletion The retinal ganglion cells are magnetically labeled with CD90.1 CD90.1 with labeled magnetically are cells ganglion retinal The The Retinal Ganglion Cell Isolation Kit, rat has been developed developed been has rat Kit, Isolation Cell Ganglion Retinal The 1.1 Applications 1.3 1.2 page 1/4 ● CD90.1+ retinal ganglion cells are retained in the column. The The column. the in retained are cells ganglion CD90.1+ retinal Depletion Biotinylated the with labeled concurrently CD90.1, are MicroBeads. Endothelial cells and microglia, which also express express also which microglia, and cells Endothelial MicroBeads. diseases like glaucoma. RGCs were among the first neurons to neurons first the were among RGCs glaucoma. like diseases system nervous central of the neurons are cells ganglion Retinal cell fraction is separated over a second column. over asecond separated is fraction cell cell The pre-enriched supernatant. the in pre-enriched are cells is microglia and cells endothelial MACSiBead-labeled of the unlabeled cells run through. After removing the column from the from column the removing After through. run cells unlabeled neuroregenerative therapies. The best period for isolation of isolation for period best The therapies. neuroregenerative magnetic field, the magnetically retained CD90.1+ cells are eluted eluted are CD90.1+ cells retained magnetically the field, magnetic morphology are observed after isolating RGCs using magnetic cell cell magnetic using RGCs isolating after observed are morphology retina. rat (RGCs) from cells ganglion of retinal isolation for the suspension is loaded onto a MACS® MS Column, which is placed in in placed is which Column, MS onto aMACS® loaded is suspension sorting⁵. as the positively selected cell fraction, and the positively selected selected positively the and fraction, cell selected positively the as and project information from the eye to the visual center of the of the center visual the to eye the from information project and the magnetic field of a MACS Separator. The magnetically labeled labeled magnetically The Separator. MACS of a field magnetic the ganglion retinal The Separator. MACSiMAG™ the using performed these neurons is postnatal days 6–7. High viability and normal normal and 6–7. viability days High postnatal is neurons these of development for the useful are RGCs research, pre-clinical been used to study interaction of neurons with glial cells³ glial with of neurons interaction study to used been have They conditions². defined under cultured and isolated¹ be ophthalmic in blindness to leads of RGCs Degeneration brain.

1. 1. 2. 3. 3. Antibody, Antibody, CD90.1 MicroBeads, with labeling Magnetic using a MACSiMAG Separator. Depletion of MACSiBead-labeled endothelial cells and microglia microglia and cells endothelial MACSiBead-labeled of Depletion or OctoMACS Separator and two MS Columns consecutively. Columns MS two and Separator OctoMACS or aMiniMACS using cells ganglion retinal of selection Positive Isolation of neurons, namely rat retinal ganglion cells. ganglion retinal rat namely of neurons, Isolation Principle of the MACS® Separation MACS® of the Principle Background information Background and Anti-Biotin MACSiBeadand Particles Anti-Biotin Single-cell suspension fromSingle-cell retinal tissue Pre-enriched retinal ganglionPre-enriched cells CD 90.1 + retinal ganglion cells Order no. 130-096-209

Biotinylated Depletion .

, ⁴. In 140-003-194.02 ▲ The use of other dissociation protocols has not been tested. tested. been not has protocols dissociation of other use The in use for developed was Kit Isolation Cell Ganglion Retinal The 1.4 2. Tissue Dissociation Kit – Postnatal Neurons (# Neurons 130-094-802). –Postnatal Kit Dissociation Tissue 2.1 ● ● ● ● ● ● ● ● ● ● ● are for research useonlyandnotfor diagnostic ortherapeutic use. combination with the Neural Tissue Dissociation Kit – Postnatal –Postnatal Kit Dissociation Tissue Neural the with combination Neurons) and ensures epitope integrity for the magnetic labeling. labeling. magnetic for the integrity epitope ensures Neurons) and (# Neurons 130-094- –Postnatal Kit Dissociation Tissue Neural 802). For a detailed protocol refer to the data sheet of the Neural Neural of the sheet data the to refer protocol 802). For adetailed Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products 1

Prepare a single-cell suspension from rat retina tissue using the the using tissue retina rat from suspension asingle-cell Prepare (Optional) Propidium Iodide Solution (# Solution Iodide Propidium (Optional) (# 130-092-197) Analyzer MACSQuant® (Optional) for flow antibodies Fluorochrome-conjugated (Optional) (# 130-092-168) Separator MACSiMAG™ Tube (# 130-090-753) Rotator MACSmix™ 130- (# Neurons –Postnatal Kit Dissociation Tissue Neural tubes bottom round polystyrene 5 mL Unit (# Separation MiniMACS™ (# Columns MS Protocol DPBS/BSA buffer: Dulbecco’s phosphate-buffered saline (DPBS) saline phosphate-buffered Dulbecco’s buffer: DPBS/BSA Reagent and instrument requirements instrument and Reagent Sample preparation Sample (# 7-AAD for flow cytometric exclusion of dead cells. dead of exclusion cytometric 7-AAD for flow CL045PE). For more information about antibodies refer to to refer antibodies about For more information CL045PE). www.miltenyibiotec.com/antibodies. Ca² with cytometric analysis, e.g., CD90.1-FITC, mouse and rat rat and mouse CD90.1-FITC, e.g., analysis, cytometric P6–7) age (20 rats, retinas 40 from derived cells 100 e.g., Separation Unit (# 130-042-109) Separation 094-802) to 5 days. Bring to room temperature before use. before temperature room to Bring 5days. to 5 mL polystyrene round bottom tubes bottom round polystyrene 5 mL 130-094-527) or CD48-PE (Cedarlane Laboratories Laboratories (Cedarlane or CD48-PE 130-094-527) + mL DPBS and 0.5 g BSA. 0.5 DPBS and mL and Mg² and 130-042-201), maximum column capacity: capacity: column 130-042-201), maximum + and with 0.5% bovine serum albumin (BSA), albumin serum bovine 0.5% with and 130-042-201) or OctoMACS™ 130-042-201) or OctoMACS™ Store buffer at 2–8 °C for up 2–8 at Store buffer 130-093-233) or

▲ ▲ 1. 10 4. 3.

▲ 2. 7. 9. 6. 5. with fewer rats is not recommended. When working with retinas retinas with working When not recommended. is rats fewer with page 2/4 page of all indicated reagent volumes and total volumes. volumes. total and volumes reagent indicated of all indicated. otherwise 8. from more rats, scale up all reagent volumes and total volumes volumes total and volumes reagent up all scale more rats, from accordingly, e.g., for 20 retinas from 10 rats, use twice the volume volume the twice use 10 from rats, retinas for 20 e.g., accordingly,

Volumes for magnetic labeling given below are for cells from from for cells are below given labeling Volumes for magnetic unless temperature at room performed be must steps All retinas (5 rats, age P6–7) in a final volume of 500 µL. Working Working 500 µL. of volume afinal P6–7) in age (5 rats, retinas 15 times using a 1 mL pipette tip. Do not vortex the cells. the not vortex Do tip. pipette a1mL using 15 times 1:160, (dilution CD48-PE 3.11:20) Cedarlane and µL Incubate for 15 minutes at room temperature using the the using temperature at room for 15 minutes Incubate Proceed to magnetic separation: Depletion of endothelial cells cells of endothelial Depletion separation: magnetic to Proceed (Optional) For flow cytometric analysis, add staining staining add analysis, cytometric For flow (Optional) Mix gently and incubate for 15 minutes at room temperature. temperature. at room for 15 minutes incubate and gently Mix of Biotinylated 10 and µL MicroBeads of CD90.1 µL Add 50 Resuspend in 400 µL of DPBS/BSA buffer by pipetting gently gently by pipetting buffer of DPBS/BSA µL 400 in Resuspend 5 by adding Wash cells Resuspend Anti-Biotin MACSiBeads Particles thoroughly thoroughly Particles MACSiBeads Anti-Biotin Resuspend (5 440 µL rats) in 10 from retinas obtained cells the Resuspend Immediately add the 400 µL of single-cell suspension and mix mix and suspension of single-cell µL 400 the add Immediately Depletion Antibody. very gently with the Anti-Biotin MACSiBeads Particles. MACSiBeads Anti-Biotin the with gently very Mix gently every 5 minutes. 5minutes. every gently Mix MACSmix Tube Rotator (permanent run, medium speed/8 speed/8 medium run, Tube (permanent Rotator MACSmix for 30 by vortexing e.g., solution, in Particles Immediately afterwards pipette 100 µL into a 5 mL tube. mL into a5 µL 100 pipette afterwards Immediately of DPBS/BSA buffer. buffer. of DPBS/BSA Laboratories) after 5 minutes of this incubation period. Mix Mix period. incubation this of minutes 5 after Laboratories) rpm). rpm). size of the 5 mL tube. 5mL the of size gently and continue incubation for the remaining 10 minutes. 10 minutes. remaining for the incubation continue and gently minutes. Aspirate supernatant carefully. supernatant Aspirate at 130×g for 5 minutes. (# CD90.1-FITC µL 25 130-094-527, dilution e.g., antibodies, and microglia (2.3). microglia and before use to obtain a homogeneous dispersion of MACSiBead of MACSiBead dispersion ahomogeneous obtain to use before

Note:

The large rack of the MACSmix MACSmix the of rack large The 2.2 Magnetic labeling Magnetic mL of DPBS/BSA buffer and centrifuge centrifuge and buffer of DPBS/BSA mL Tube Rotator can be adjusted to the the to adjusted be can Tube Rotator Order no. 130-096-209 no. Order seconds. seconds.

140-003-194.02 ▲ ▲ 1. 12. 11. 10. 4. 3. ▲ ▲ 2. 7. 9. 6. 5. are for research useonlyandnotfor diagnostic ortherapeutic use. 8. Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Do not touch the adhered cells when pipetting off the supernatant. the off pipetting when cells adhered the not touch Do Separator. aMACSiMAG Choose

Aspirate supernatant carefully. supernatant Aspirate Retaining the tube in the MACSiMAG Separator, carefully carefully Separator, MACSiMAG the in tube the Retaining Place 5 mL tube containing the pre-enriched fraction of fraction pre-enriched the containing tube 5mL Place Proceed to magnetic separation: Positive selection of CD90.1 selection Positive separation: magnetic to Proceed Wash the adhered cells by rinsing the wall of the tube in the the in tube of the wall the by rinsing cells adhered Wash the carefully Separator, MACSiMAG the in tube the Retaining MACSiMAG the of field magnetic the in tube 5mL Place Allow the MACSiBead-labeled cells to adhere to the wall of the of the wall the to adhere to cells MACSiBead-labeled the Allow Remove the 5 mL tube from MACSmix Tube open and Rotator MACSmix from tube 5 mL the Remove Resuspend cells carefully in 500 µL of DPBS/BSA buffer by buffer of DPBS/BSA µL 500 in carefully cells Resuspend temperature. at room at 130×g for 2minutes Centrifuge carefully Separator, MACSiMAG the in tube the Retaining for one minute. wall tube the to adhere to cells the Allow vortex the cells. the vortex MACSiBead-labeled cells to adhere to the tube wall for 3 wall tube the to adhere to cells MACSiBead-labeled of DPBS/BSA. mL 0.5 with Separator MACSiMAG containing the retinal ganglion cells. ganglion retinal the containing retinal ganglion cells (2.4). cells ganglion retinal see the MACSiMAG Separator data sheet. data Separator MACSiMAG the see minutes. 4. of step fraction remaining target cells will be collected in the next step. next the in collected be will cells target remaining Separator. step 7 into the MACSiMAG Separator. Allow remaining remaining Allow Separator. MACSiMAG the 7into step pipetting gently 15 times using a 1 mL pipette tip. Do not Do tip. pipette a1mL using 15 times gently pipetting tube. a15 into mL fraction) (pre-enriched supernatant the pipette pre-enriched the containing tube the into supernatant pipette fraction the pre-enriched is supernatant The magnet. the of outside placed tube 5mL anew into supernatant pipette for 3 minutes. tube in the tube. the in µL 500 it the to add and of DPBS/BSA µL 300 with lid Rinse it.

Note: Note: Note: 2.3

In this step, the supernatant does not need to be completely removed; removed; completely be to need not does supernatant the step, this In Use tube rack to insert tubes from 1.5 mL to 5 mL in size. For details details For size. in 5mL to mL 1.5 from tubes insert to rack tube Use endothelial cells and microglia and cells endothelial Magnetic separation: Depletion of Depletion separation: Magnetic

▲ ▲ ▲ ▲ 1. 1.4. 4. 3. ▲ 2. 7. 9. 6. 5. P6–7) can be applied to an MS Column. Column. MS an to applied be P6–7) can page 3/4 page column reservoir is empty. is reservoir column 8.

Perform Perform consecutively. Columns 2MS using separated are Samples (age rats or 20 retinas up40 to from derived of cells A maximum section see For details Separator. MACS appropriate an Choose Analyzer. Cultivate cells in a poly-L-lysine-coated dish in in dish apoly-L-lysine-coated in cells Cultivate Analyzer. 3×500 µL of DPBS/BSA buffer. of DPBS/BSA 3×500 µL Determine cell number by using, for example, the MACSQuant MACSQuant the for example, by using, number cell Determine Pipette 1 mL of DPBS/BSA buffer onto the column. column. the onto buffer of DPBS/BSA 1 mL Pipette Apply cell suspension onto the first MS Column. Collect Collect Column. MS first onto the suspension Apply cell buffer. of DPBS/BSA 500 µL with by rinsing columns Prepare Remove first column from the separator and hold it over the and hold it over separator the from column first Remove Collect buffer. of DPBS/BSA µL 3×500 with Wash column Let cells pass through the second MS Column and wash with with wash and Column MS second the through pass cells Let Place 2 MS Columns in the magnetic field of a suitable MACS suitable of a field magnetic the in Columns 2MS Place Remove second MS Column from the separator and place it place and separator the from Column MS second Remove flow-through containing unlabeled cells in case analysis is of analysis case in cells unlabeled containing flow-through will not harm the cells. the harm not will Immediately flush out the magnetically labeled cells by firmly firmly by cells labeled magnetically the out flush Immediately onto a suitable collection tube. collection onto suitable a unlabeled cells that pass through and combine with flow- with combine and through pass that cells unlabeled magnetically labeled cells by firmly pushing the plunger into the plunger pushing by firmly cells labeled magnetically second equilibrated MS Column. Pipette 1 mL of DPBS/BSA of DPBS/BSA 1 mL Pipette Column. MS equilibrated second sheet. data Column MS the to refer For details Separator. subsequent applications. subsequent an appropriate density at 37 density appropriate an the column. the analysis. 3for optional step from through pushing the plunger into the column. the into plunger the pushing interest. buffer onto the first MS Column and immediately flush out the out flush immediately and Column MS first the onto buffer

Note: 2.4

washing steps by adding buffer aliquots as soon as the as soon as aliquots buffer by adding steps washing Unbound MACSiBead Particles may be observed in cell culture but but culture cell in observed be may Particles MACSiBead Unbound CD90.1 Magnetic separation: Positive selection of selection Positive separation: Magnetic + retinal ganglion cells ganglion retinal °C or proceed immediately to to immediately or proceed °C Order no. 130-096-209 no. Order 140-003-194.02 References4. 1. 3. 4. fluorescently stained with CD48-PE and CD90.1-FITC CD90.1-FITC and (# CD48-PE with stained fluorescently are for research useonlyandnotfor diagnostic ortherapeutic use. Miltenyi Biotec provides technical support worldwide. Visit worldwide. support technical provides Biotec Miltenyi www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec Biotec Miltenyi nearest your find to www.miltenyibiotec.com/local 2. Refer to to Refer Isolation of CD90.1 of Isolation contact. cells were excluded from the analysis based on scatter signals and and signals on scatter based analysis the from were excluded cells Neurons, the Retinal Ganglion Cell Isolation Kit, a MACSiMAG aMACSiMAG Kit, Isolation Cell Ganglion Retinal the Neurons, 3. retina tissue using the Neural Tissue Dissociation Kit - Postnatal -Postnatal Kit Dissociation Tissue Neural the using tissue retina 6. Separator, an OctoMACS Separator, and MS Columns. Cells were Cells Columns. MS and Separator, OctoMACS an Separator, 5. 094-527) and analyzed by flow cytometry. Cell debris and dead dead and debris Cell cytometry. by flow analyzed and 094-527) propidium iodide fluorescence. iodide propidium Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products Vol. 12-2/2010. Vol. Example of a separation using the the using of aseparation Example Meyer-Franke, A. A. Meyer-Franke, Göritz, C. C. Göritz, Pfrieger, F. W. and Barres, B. A. (1997) Synaptic efficacy enhanced by glial cells cells glial by enhanced efficacy (1997) Synaptic A. B. F. W. Barres, and Pfrieger, Bottenstein, J. E. and Sato, G. H. (1979) Growth of a rat neuroblastoma cell line line cell neuroblastoma arat of (1979) H. G. Growth Sato, and E. J. Bottenstein, A. A. B. Barres, Pennartz, S Pennartz, culture. Neuron 15: 805–819. 15: Neuron culture. Retinal Ganglion Ganglion Retinal electrophysiological variation among retinal ganglion cells purified by purified cells ganglion retinal among variation electrophysiological panning.Neuron 1: 791–803. panning.Neuron in vitro in regulation. Glia 55: 1108–1122. 55: Glia regulation. that promote the survival and growth of developing retinal ganglion cells in in cells ganglion retinal developing of growth and survival the promote that in serum-free supplemented medium. Proc. Natl. Acad. Sci. U.S.A. 76: 514–517. U.S.A. Sci. Acad. Natl. Proc. medium. supplemented serum-free in www.miltenyibiotec.com . Science 277: 1684–1687. 277: Science . et al. et et al. et (2007) Glia-induced neuronal differentiation by transcriptional by transcriptional differentiation neuronal Glia-induced (2007) (2010) Purification of retinal ganglion cells. MACS&more MACS&more cells. ganglion retinal of (2010) Purification et al. et et al. et + CD90.1 retinal ganglion cells from postnatal day 7 rat 7rat day postnatal from cells ganglion retinal CD90.1-FIT C CD90.1-FIT C (1995) Characterization of the signaling interactions interactions signaling the of (1995) Characterization (1988) Immunological, morphological, and and morphological, (1988) Immunological, Before separation Before Cell Isolation Kit Cell + retinal ganglion cells ganglion retinal CD48-PE CD48-PE for all data sheets and protocols. protocols. and sheets data for all

130-

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