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Errata

Erratum: One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

doi: 10.3791/5614 Published: November 26, 2014
TODO, TODO

Abstract

A correction was made to One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice. Many micro symbols were changed into milli symbols by accident: In the Protocols, sections 1.1, 1.3, 3.6, 3.7, 6.2, 6.6, 8.1, 9.1.6, 9.2.2, and 9.2.5 need to be fixed, as does Figure 1 description in the Results section.

Protocol section 1.1 was changed from:

At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 mg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 mg/ml in cold DMEM:F12) and incubate at 37 °C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 °C until required.

to:

At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 µg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 µg/ml in cold DMEM:F12) and incubate at 37 °C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 °C until required.

Protocol section 1.3 was changed from:

On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 µg/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 °C in a water bath.

to:

On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 mg/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 °C in a water bath.

Protocol section 3.6 was changed from:

Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 mm sieve to remove debris and undissociated tissue clumps.

to:

Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 µm sieve to remove debris and undissociated tissue clumps.

Protocol section 3.7 was changed from:

Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 ml growth medium.

to:

Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 µl growth medium.

Protocol section 6.2 was changed from:

Add 50 ml Accutase and incubate at 37 °C for 2-3 min (checking to see if the cells are rounded and detached).

to:

Add 50 µl Accutase and incubate at 37 °C for 2-3 min (checking to see if the cells are rounded and detached).

Protocol section 6.6 was changed from:

For subsequent passages, resuspend cells in 200 ml growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.

to:

For subsequent passages, resuspend cells in 200 µl growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.

Protocol section 8.1 was changed from:

Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 ml/well across a 96-well plate using a 10 ml multidoser pipette.

to:

Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 µl/well across a 96-well plate using a 10 ml multidoser pipette.

Protocol section 9.1.6 was changed from:

Remove 10 ml of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.

to:

Remove 10 µl of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.

Protocol section 9.2.2 was changed from:

Add 100 ml of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.

to:

Add 100 µl of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.

Protocol section 9.2.5 was changed from:

Transfer the 200 ml containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 °C with 5% CO2 until secondary neurospheres form.

to:

Transfer the 200 µl containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 °C with 5% CO2 until secondary neurospheres form.

Figure 1 description was updated from:

Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 mm.

to:

Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 µm.

Protocol

A correction was made to One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice. Many micro symbols were changed into milli symbols by accident: In the Protocols, sections 1.1, 1.3, 3.6, 3.7, 6.2, 6.6, 8.1, 9.1.6, 9.2.2, and 9.2.5 need to be fixed, as does Figure 1 description in the Results section.

Protocol section 1.1 was changed from:

At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 mg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 mg/ml in cold DMEM:F12) and incubate at 37 °C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 °C until required.

to:

At least two days prior to commencing the experiment, prepare Poly-D-lysine (PDL)/Laminin coated plates for adherent monolayer cultures. To prepare wells/flasks add enough PDL (10 µg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add Laminin (5 µg/ml in cold DMEM:F12) and incubate at 37 °C overnight. Remove the Laminin and either use the plates immediately or store with the Laminin at -20 °C until required.

Protocol section 1.3 was changed from:

On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 µg/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 °C in a water bath.

to:

On the day of dissection, prepare the appropriate amount of culture medium by mixing Neural Basal Medium with 2% B27, 1x GlutaMAX, 2 mg/ml heparin, 50 units/ml Penicillin/Streptomycin, 20 ng/ml purified mouse receptor-grade epidermal growth factor (EGF), and 20 ng/ml recombinant bovine fibroblast growth factor (FGF-2). Warm the culture medium to 37 °C in a water bath.

Protocol section 3.6 was changed from:

Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 mm sieve to remove debris and undissociated tissue clumps.

to:

Add growth medium to a total volume of 5 ml and pass the cell suspension through a 40 µm sieve to remove debris and undissociated tissue clumps.

Protocol section 3.7 was changed from:

Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 ml growth medium.

to:

Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the resulting pellet in 200 µl growth medium.

Protocol section 6.2 was changed from:

Add 50 ml Accutase and incubate at 37 °C for 2-3 min (checking to see if the cells are rounded and detached).

to:

Add 50 µl Accutase and incubate at 37 °C for 2-3 min (checking to see if the cells are rounded and detached).

Protocol section 6.6 was changed from:

For subsequent passages, resuspend cells in 200 ml growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.

to:

For subsequent passages, resuspend cells in 200 µl growth medium and count using a hemocytometer. Plate at 1 x 104 cells/cm2 in the appropriate sized coated well or flask.

Protocol section 8.1 was changed from:

Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 ml/well across a 96-well plate using a 10 ml multidoser pipette.

to:

Dilute the dissociated SVZ or DG tissue from one animal in 20 ml of culture medium and plate 200 µl/well across a 96-well plate using a 10 ml multidoser pipette.

Protocol section 9.1.6 was changed from:

Remove 10 ml of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.

to:

Remove 10 µl of the cell suspension and mix with an equal volume of trypan blue and perform a live cell count using a hemocytometer.

Protocol section 9.2.2 was changed from:

Add 100 ml of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.

to:

Add 100 µl of 0.05% Trypsin-EDTA to each well to be passaged and incubate at room temperature for 3 min.

Protocol section 9.2.5 was changed from:

Transfer the 200 ml containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 °C with 5% CO2 until secondary neurospheres form.

to:

Transfer the 200 µl containing the dissociated neurosphere to a new well of a 24-well plate containing 1.5 ml of growth medium. Incubate at 37 °C with 5% CO2 until secondary neurospheres form.

Figure 1 description was updated from:

Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 mm.

to:

Figure 1. Adult mouse precursor cells can be cultured as adherent monolayer cultures (A) or as neurospheres (B: SVZ, C: DG). Scale bar is 50 µm.

Disclosures

No conflicts of interest declared.

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Cite this Article

TODO, T. Erratum: One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice. J. Vis. Exp. (93), e5614, (2014).More

TODO, T. Erratum: One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice. J. Vis. Exp. (93), e5614, (2014).

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