• ioOligodendrocyte-like cells (io1028) cultured in a 24 well plate following the user manual at a seeding density of 27,000 cells/cm². Refer to the latest user manual for optimal culturing conditions.

• Biological safety cabinet with a carbon filter (MSC-CF)

• Normoxic cell culture incubator (37°C, 5% CO2)

• -80°C freezer

• 1000 μL, 200 μL, 20 μL and 10 μL micropipettes

• Standard light microscope

• Epifluorescent microscope

• 1.5 mL microcentrifuge tubes (Starlab, S1615 5510)

• 15 mL centrifuge tube, conical (Greiner Bio-one, 188271)

• 50 mL centrifuge tube, conical (Greiner Bio-one, 227261)

• 24 well plate TC-treated (Scientific Laboratory Supplies, 3526)

• DPBS, no calcium, no magnesium (ThermoFisher, 14190144)

• 16% paraformaldehyde (ThermoFisher, 11586711)

• Saponin (Sigma-Aldrich, 47036-50G-F)

• Goat serum (Sigma-Aldrich, G9023-10ML)

• Parafilm (Fisher Scientific, 10018130)

This protocol is split into 4 sections:

1: Cell fixation

2: Blocking and permeabilization

3: Primary antibody labelling

4: Secondary antibody labelling

1. Cell fixation

1.1. Prepare fixation solution (4% paraformaldehyde/DPBS) as described in Appendix 1.

1.2. Slowly remove the culture medium without disturbing the cells.

1.3. Carefully add 200 µL of DPBS by pipetting down the side of the wells.

1.4. Gently remove the DPBS without disturbing the cells.

1.5. Carefully add 200 µL of room temperature fixation solution. 

1.6. Incubate for 10 minutes at room temperature.

1.7. Gently remove the fixation solution without disturbing the cells.

1.8. Carefully add 200 μL of DPBS to each well.

1.9. Gently remove the DPBS without disturbing the cells.

1.10. Add 500 μL of DPBS per well, wrap the plate with parafilm and store at 4°C overnight or until staining. Staining should be done ideally within 14 days of fixation.

2. Permeabilization and blocking

2.1. Prepare the following reagents as described in Appendix 1: 

2.1.1 Permeabilization solution (0.02% Saponin in DPBS).

2.1.2. Blocking solution (10% Normal Goat Serum in DBPS).

2.2. Gently remove the DPBS without disturbing the cells.

2.3. Carefully add 200 µL of permeabilization solution to each well.

2.4. Incubate for 15 minutes at room temperature.

2.5. Gently remove the permeabilization solution without disturbing the cells.

2.6. Carefully add 200 µL of blocking solution to each well.

2.7. Incubate for 30 minutes at room temperature.

3. Primary antibody labelling

3.1. Select one test well and one control well from each of your conditions and timepoints.

3.2. Prepare the primary buffer (0.02% saponin / 2% normal goat serum in DPBS) and primary antibody mixture (0.02% saponin / 2% normal goat serum in DPBS + primary antibodies) described in Appendix 1.

3.3. Following the incubation (described in step 2.7), gently remove the blocking solution using a micropipette.

3.4. Carefully add 200 μL of primary antibody mixture by pipetting down the side of the test wells.

3.5. Carefully add 200 μL primary buffer to the control wells.

3.6. Seal plates with parafilm and incubate overnight at 4°C.

3.7. Following the incubation, gently remove the solution without disturbing the cells.

3.8. Carefully add 250 μL of DPBS to each well.

3.9. Incubate for 5 minutes at room temperature.

3.10. Repeat steps 3.7 to 3.9 a further 2 times, for a total of 3 wash steps, leaving the cells in 250 μL of DPBS before moving on to secondary antibody labelling.

4. Secondary antibody labelling

4.1. Prepare the secondary antibody mixture (1% normal goat serum in DPBS + DAPI + secondary antibodies) described in Appendix 1.

4.2. Gently remove the DPBS without disturbing the cells.

4.3. Carefully add 200 μL of secondary antibody mixture by pipetting down the side of the wells.

4.4. Incubate for 1 hour at room temperature.

4.5. Following the incubation, gently remove the solution from each well without disturbing the cells.

4.6. Carefully add 250 μL of DPBS to each well.

4.7. Incubate cells for 5 minutes at room temperature in the dark.

4.8. Repeat steps 4.5 to 4.7 a further 3 times, for a total of 4 wash steps, leaving the cells in 500 μL of DPBS.

4.9. Image each well using a fluorescent microscope with the fluorescent channel most appropriate for each antibody.

Fixation solution (4% paraformaldehyde/DPBS):

1. Add 30 mL DPBS to a 50 mL centrifuge tube.

2. Add 10 mL of 16% paraformaldehyde.

3. Allow the fixation solution to warm to room temperature before use.

Permeabilization solution (0.02% saponin in DPBS):

1. To prepare a 5% saponin solution in DPBS:

1.1. Weigh 1 g of saponin powder into a 50 mL falcon tube.

1.2. Add 20 mL DPBS.

1.3. Vortex vigorously to fully dissolve the saponin.

2. To prepare permeabilization solution (0.02% saponin in DPBS):

2.1 Add 80 μL of 5% saponin solution to 20 mL of fresh DPBS and vortex vigorously.

Blocking solution (10% normal goat serum in DBPS):

1. Mix 500 μL goat serum with 4.5 mL DPBS.

Primary buffer (0.02% saponin / 2% normal goat serum in DPBS):

1. Mix 10 μL of goat serum with 472.5 μL of permeabilization solution (0.02% saponin in DPBS).

Primary antibody mixture (0.02% saponin / 2% normal goat serum in DPBS + primary antibodies):

1. Centrifuge the stock primary antibody tubes for 5 seconds using a mini centrifuge.

2. Dilute antibodies in the primary buffer according to the recommended dilution described in appendix 2.

Secondary antibody mixture (1% normal goat serum in DPBS + DAPI + secondary antibodies):

1. Add 10 μL goat serum to 1 mL DPBS.

2. Add 2 μL of DAPI (1 mg/mL stock concentration, for a final 2 μg/mL concentration) to the final 1 mL solution.

3. Centrifuge the stock secondary antibody tubes for 5 seconds using a mini centrifuge.

4. Dilute secondary antibodies according to the recommended dilution described in appendix 2.

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If you have any questions or need assistance, please reach out to technical@bit.bio and we will do our best to support you.

Published February 2024, version 1.