Column | Max. number of labeled cells | Max. number of total cells | Separator |
---|---|---|---|
Positive selection | |||
MS | 1×10⁷ | 2×10⁷ | MiniMACS™, OctoMACS™, VarioMACS, SuperMACS II |
LS | 2×10⁷ | 4×10⁷ | MidiMACS™, QuadroMACS, VarioMACS, SuperMACS II |
Depletion | |||
LD | 1.5×10⁷ | 3×10⁷ | MidiMACS, QuadroMACS, VarioMACS, SuperMACS II |
Positive selection or depletion | |||
autoMACS | 5×10⁷ | 1×108 | autoMACS Pro |
DPBS/BSA buffer: Prepare a solution containing Dulbecco’s phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with DPBS. Keep buffer cold (2–8 °C). Degas buffer before use, as air bubbles could block the column.
▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).
Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.
Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.
Use the Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.
Download data sheet
Isolate CD171 (L1CAM)-positive neurons from the single-cell suspension using the CD171 (L1CAM) MicroBead Kit. Follow the protocol of the kit data sheet.
Download data sheet
To stain and analyze the isolated neurons by flow cytometry, follow the protocol on cell surface flow cytometry staining.
Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse (1:10).
Primary Antibody | Secondary Antibody | |||||
---|---|---|---|---|---|---|
Antibody | Incubation temperature | Incubation time | Recommended antibody concentration | Secondary antibody | Incubation temperature | Incubation time |
Anti-ACSA-2 | Room temperature | 10 minutes | 1–5 µg/mL | Anti-rat IgG2b | Room temperature | 10 minutes |
Anti-GLAST (ACSA-1) | Room temperature | 10 minutes | 1–5 µg/mL | Anti-mouse IgG2a | Room temperature | 10 minutes |
Anti-O4 | Room temperature | 10 minutes | 5–10 µg/mL | Anti-mouse IgM | Room temperature | 10 minutes |
Anti-PSA-NCAM | Room temperature | 10 minutes | 5–10 µg/mL | Anti-mouse IgM | Room temperature | 10 minutes |
CD11b | Room temperature | 10 minutes | 5–10 µg/mL | Anti-rat IgG2bκ | Room temperature | 10 minutes |
CD68 | 2–8 °C | Overnight | 5–10 µg/mL | Anti-rat IgG2a | Room temperature | 1 hour |
CD171 (L1CAM) | 2–8 °C | Overnight | 5–10 µg/mL | Anti-rat IgG2a | Room temperature | 1 hour |
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autoMACS, CliniMACS, the CliniMACS logo, CliniMACS Prodigy, CryoMACS, CytoMix, CytoStim, DendriMACS, ExiTron, ExpAct, FeraSpin, FeraTrack, GadoSpin, gentleMACS, LIFE 18, LIFE 21, MACS, the MACS logo, MACSductin, MACSelect, MACSfectin, MACSflex, MACSiBead, MACSiMAG, MACSmix, MACSprep, MACSQuant, MACSQuantify, MACSxpress, MidiMACS, MiniMACS, miRXplore, MultiMACS, NeuroBrew, NiraWave, OctoMACS, PepTivator, pMACS, PolySon, PrepProtect, QuadroMACS, REAfinity, REAlease, Rheo, StemMACS, StraightFrom, SuperAmp, SuperMACS, TexMACS, TheraSorb, thermoMACS, TransAct, Tyto, the Tyto logo, VarioMACS, Vio, Viobility, VioBlue, VioBright, VioGreen, Viscover, and µMACS are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide. Ficoll-Paque is a trademark of GE Healthcare companies. Vectofusin-1 is a registered trademark of Genethon. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec B.V. & Co. KG and/or its affiliates. All rights reserved.
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