Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

​All animal protocols used in this study were approved by the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University and performed in accordance with guidelines of the IACUC. The laboratory animal facility at Tsinghua University has been accredited by the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). For staging of embryos, mid-day of the vaginal plug identified was calculated as embryonic day 0.5 (E0.5).

NOTE: All cells are cultured in the cell incubator under conditions of 37 °C and 5% CO₂.

1. Preparation of Mouse Endothelial Culture in Permeable Support Inserts

NOTE: BEND3 cells are maintained according to manufacturer's instructions.

1. Prepare BEND3 cell medium (BM) by adding 50 mL of FBS and 5 mL of penicillin-streptomycin into 500 mL of DMEM and mix well.
2. Aspirate the medium from the dish and wash the BEND3 cells culture with 1 mL of PBS once. Add 1 mL of 0.25% Trypsin-EDTA into the cells and incubate the cells for 4 min at 37 °C.
3. Add 1 mL of BM into the cells to neutralize Trypsin-EDTA and pipette up and down gently to completely dissociate the cells. Transfer the cell suspension into a new 15 mL conical tube and pellet by centrifugation at room temperature (RT) for 5 min at 400 x g.
4. Aspirate the supernatant from the tube and resuspend the cells with 9 mL of fresh BM, then add 1 mL of cell suspension into one permeable support insert. Add another 2 mL of fresh BM per well at the bottom chamber of the matrix. Continue to culture the cells for one day.

2. Preparation of Mouse Primary Cortical NSPCs Culture

1. Preparation of culture plate, papain digestion medium, and cortical adherent culture medium (AM)
   1. Coat 6-well plates with poly-L-lysine (PLL) by adding 1 mL of PLL solution per well into 6-well plates. Then, incubate the plates at RT for 30 min.
   2. Transfer the PLL solution into a 15 mL conical tube. Wash the plates 3 times with double distilled water. Airdry the plates and put them aside until use.
   3. Prepare the papain digestion medium by adding 50 U of papain, 50 µL of L-glutamine, and 50 µL of 100 mg/mL acetyl-L-cysteine into 5 mL of DMEM. Mix the medium briefly and warm it to 37 °C for 30 min for enzyme activation.
   4. Prepare the cortical cell adherent culture medium (AM): add 500 µL of L-glutamine, 500 µL of sodium pyruvate, 500 µL of 100 mg/mL N-acetyl-L-Cysteine, 500 µL of N2, 1 mL of B27, and 5 µL of 100 µg/mL bFGF into 50 mL of DMEM. Mix the medium well and warm it to 37 °C before use.
2. Preparation of primary cerebral cortical cells and subsequent plating
   1. Sacrifice an E10.5 timed pregnant mouse by cervical dislocation.
      NOTE: At E10.5, a majority of cells are proliferating NSPCs in the cerebral cortex, giving rise to large clones of progeny in vitro.
   2. Sterilize the abdomen by 75% ethanol. Use fine scissors and micro-serrated forceps to open the abdomen by cutting the skin and underlying muscle along the right side of the middle line. Remove the uterus from the abdominal cavity gently with serrated forceps and cut it out from the abdominal cavity with fine scissors.
   3. Wash the uterus with 40 mL of pre-chilled HBSS in 10 cm Petri dish. Then, transfer the uterus into a new 10 cm Petri dish and wash it again with 40 mL of pre-chilled HBSS.
   4. Transfer the uterus into a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Remove the embryos from the uterus and amniotic membrane, then cut the heads of the embryos off from the trunks with Jewelers microforceps.
   5. Wash the heads with 40 mL of pre-chilled HBSS and transfer the heads to a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Use Jewelers microforceps to peel away skin and cartilage covering the brains, then cut the cerebral cortices off and collect them in a 15 mL conical tube with pre-chilled HBSS.
   6. Pellet the cortices by centrifugation for 3 min at 4 °C and 300 x g. Aspirate the supernatant from the tube, then add activated papain digestion medium and 15 µL of 4 mg/mL DNase I into the tissue pellet.
   7. Resuspend the tissue pellet briefly by gentle vortexing. Incubate the tissue at 37 °C for 30 min. During this time, loosen the tissue by brief vortexing every 10 min.
      NOTE: At the end of the digestion, there should be no visible tissue pieces in the tube.
   8. Pellet the cortical cells by centrifugation for 10 min at 4 °C and 450 x g. Aspirate the supernatant from the tube and wash the cell pellet with pre-chilled DMEM. Repeat this step once.
      NOTE: During the digestion and washing, take caution not to pipette the tissues and cell pellet roughly to avoid damaging the cells with a strong shearing force.
   9. Aspirate the supernatant from the tube then add 1.5 mL of pre-chilled HBSS into the tube. Dissociate the cortical cell pellet into single cells with gentle pipetting. Count the cell number with a hemocytometer.
   10. Add 2 mL of AM and 2 x 10⁴ cortical cells per well into 6-well plates. Incubate the plate at 37 °C and 5% CO₂ for 3 h to let the cells attach to the plate.

3. Set-up of Neural-endothelial Co-culture and Ac₄ManNAz Labeling System

1. One day after plating BEND3 cells in the inserts, gently aspirate the medium in the bottom chamber first, then the inserts. Wash the enface of the inserts 3 times with pre-warmed DMEM. Wash the outer surface of the inserts by rinsing with pre-warmed DMEM.
2. Add 1 mL of pre-warmed AM into one insert, then transfer the inserts into the wells with primary cortical cells. Incubate the co-culture at 37 °C and 5% CO₂ for 12 h.
3. Dissolve Ac₄ManNAz in DMSO to achieve a stock concentration of 200 mM. 12 h after setting up the neural-endothelial co-culture, add 1 µL of Ac₄ManNAz stock per bottom chamber and 0.5 µL of stock per insert into the co-culture. Shake the plates immediately and gently to mix the medium well. For the control cells, add equal volume of DMSO.
4. Culture the cells for another 5 days at 37 °C and 5% CO₂. Prepare the AM with 10x bFGF as refeeding medium (RM). During this time, add 100 µL of RM per insert and 200 µL of RM per bottom chamber to refeed the endothelial and neural cells every other day. During the refeeding, do not supply Ac₄ManNAz or DMSO into the culture.

4. Immunofluorescent Staining of Sialoglycoproteins in Expanded Primary NSPCs and Differentiated Neurons

1. Prepare BTTAA-CuSO₄ complex 1 30x stock containing 1.5 mM CuSO₄ and 9 mM BTTAA in double-distilled water. Prepare freshly biotin-conjugated buffer 1 containing 50 µM biotin-alkyne, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO₄ complex in PBS.
2. Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-warmed PBS.
3. Aspirate the PBS from the wells. Add 1 mL of pre-chilled 4% paraformaldehyde PBS solution per well into the cells and fix the cells at RT for 10 min. Then, wash the cells 3 times with pre-chilled PBS.
4. Aspirate PBS from the wells and add 1 mL of freshly prepared biotin-conjugated buffer 1 per well into the cells. Incubate the cells at RT for 10 min.
5. Aspirate the reaction buffer from the wells. Wash the cells 3 times with PBS. Prepare the staining buffer containing 1% FBS and 1 µg/mL Alexa Fluor 647-streptavidin. Add 1 mL of staining buffer per well into the cells and incubate the cells at RT for 30 min.
6. Aspirate the staining buffer from the wells and washed cells 3 times with pre-chilled PBS. Prepare the blocking buffer containing 5% BSA and 0.3% non-ionic detergent-100 in PBS. Add 1 mL of blocking buffer per well into the cells and incubate at RT for 10 min.
7. Prepare a primary antibody solution by diluting the anti-nestin and anti-β-tubulin III antibodies together into the blocking buffer at ratios of 1:20 and 1:1,000, respectively. Remove the blocking buffer from the wells and add 1 mL of primary antibody solution per well into the cells. Incubate the cells at 4 °C overnight.
8. Remove the primary antibody solution from the wells. Wash the cells 3 times with pre-chilled PBS. Prepare a secondary antibody solution by diluting Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor 546 goat anti-mouse IgG2b, and DAPI together into blocking buffer at a dilution of 1:1,000. Aspirate the PBS from the wells and add 1 mL secondary antibody solution per well into cells. Incubate the cells at RT for 2 h.
9. Aspirate the antibody solution from the wells and wash the cells 3 times with pre-chilled PBS. Afterwards, the cells are ready for image capture.

5. Purification of Sialoglycoproteins from Expanded Primary NSPCs and Differentiated Neurons

1. Prepare BTTAA-CuSO₄ complex 2 15x stock containing 1.5 mM CuSO₄ and 3 mM BTTAA in double distilled water. Prepare protein resuspension buffer A containing 4% SDS and 10 mM EDTA in double distilled water; protein resuspension buffer B containing 150 mM NaCl, 50 mM triethanolamine, and 1% polyoxyethylene oleyl ether (e.g., Brij97) in double distilled water with pH 7.4. Before use, mix buffer A:buffer B = 1:8 (vol/vol) to prepare the full protein resuspension buffer. Prepare protein washing buffer 1 containing 2% SDS in PBS; protein washing buffer 2 containing 8 M urea in 250 mM ammonium bicarbonate (ABC); and protein washing buffer 3 containing 2.5 M NaCl in PBS.
2. Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-chilled PBS.
3. Aspirate the PBS from the wells and add 200 µL of pre-chilled RIPA buffer per well into the plates. Incubate the plates on ice for 5 min. Collect the protein lysis into 1.5 mL tubes. Pellet the cell debris by centrifugation for 10 min at 4 °C and 12,000 x g.
4. Transfer the supernatant into new 1.5 mL tubes. Determine protein concentration with the BCA kit according to the manufacturer's instructions. Adjust the protein concentration to 1 mg/mL.
5. Add 100 µM alkyne-biotin, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO₄ complex 2 to 1 mL of protein lysis and mix the solution well. Incubate the mix at RT for 1 h.
6. Transfer the reaction solution into 20 mL of pre-chilled methanol in a 50 mL conical tube. Mix well and incubate at -30 °C overnight to precipitate the proteins.
7. Pellet the protein precipitates by centrifugation for 15 min at 4 °C and 4,500 x g. Wash the protein pellet twice with 20 mL of pre-chilled methanol. Aspirate the supernatant from the tube. Resuspend the protein pellet with 4 mL of protein resuspension buffer and transfer the protein resuspension into a new 15 mL conical tube.
8. Take 50 µL of streptavidin beads and wash them 3 times with PBS. Add the washed beads into the protein resuspension. Incubate the solution at 4 °C for 3 h on a vertical rotator at a rotation speed of 20 rpm.
9. Wash the beads sequentially with 6 types of buffers: protein washing buffer 1, protein washing buffer 2, protein washing buffer 3, 0.5 M ABC, 0.25 M ABC and 0.05 M ABC.
10. After washing, resuspend the beads with 20 µL of PBS and transfer the beads into a new 1.5 mL tube. Add 20 µL of 2x protein loading buffer into the beads and treat at 95 °C for 10 min. The protein samples should then be subjected to SDS-PAGE and stained with Coomassie brilliant blue R-250 according to the manufacturer's instructions. Cut the proteins in gel as indicated by Coomassie brilliant blue R-250 for mass spectrometry analysis.