Notes:

• For subsequent cell separation and cultivation it is recommended to dissociate at least 800 mg of adult mouse or rat brain tissue. 
• Volumes given below are for one adult mouse brain (max. 500 mg) in 1980 μL enzyme mix. When working with less than 500 mg, use the same volumes as indicated. When working with higher tissue quantitities, scale up all reagent volumes and total volumes accordingly. A maximum of 500 mg brain tissue per C Tube can be processed.
• A swinging bucket rotor is recommended for centrifugation,e. g., Heraeus® Multifuge 4KR by Thermo Fisher® Scientific.

1. Enzyme P is ready to use. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months. Resuspend the lyophilized powder in the vial labeled Enzyme A with 1 mL Buffer A. Do not vortex. This solution should be aliquoted and stored at –20 °C for later use. Avoid repeated freeze-thaw-cycles.
   
2. Prepare enzyme mix 1 and enzyme mix 2 according to the table below.
   

1. Dilute the Red Blood Cell Removal Solution (10×) 1:10 with double-distilled water (ddH₂O), for example, dilute 0.1 mL of cold Red Blood Cell Removal Solution (10×) with 0.9 mL cold ddH₂O.
   ▲Note: Do not use deionized water for dilution.
2. Store the prepared 1× Red Blood Cell Removal Solution at 2–8 °C. Discard unused solution at the end of the day.

1. Prepare the following medium: DMEM containing 10% FBS, 1% penicillin/streptomycin, and 2mM L-glutamine.
2. Coat the culture dish (24-well plate) with 0.01% Poly-L-Lysine overnight at 37 °C and wash three times with ddH₂O afterwards.
   

Notes:

• For details on the use of the gentleMACS™ Octo Dissociator with Heaters, refer to the user manual.
• A maximum of 500 mg mouse or rat brain tissue in 2 mL enzyme mix can be processed in one C Tube.
• For cell culture experiments subsequent to tissue dissociation, all steps should be performed under sterile conditions.

1. Remove the mouse or rat brain. Wash the brain in cold D-PBS.
   
2. Prepare the appropriate volume of enzyme mix 1 (see above) and transfer it into a gentleMACS C Tube.
3. Place the brain on a petri dish and cut it into 8 sagittal slices using a scalpel.
4. Transfer the tissue pieces into the C Tube containing 1950 μL of enzyme mix 1.
5. Transfer 30 μL of enzyme mix 2 into the C Tube.
6. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Octo Dissociator with Heaters.
7. Run the gentleMACS Program 37C_ABDK_01.
8. After termination of the program, detach C Tube from the gentleMACS Octo Dissociator with Heaters.
9. (Optional) Centrifuge briefly to collect the sample at the bottom of the tube.
10. Resuspend sample and apply it to a MACS® SmartStrainer (70 μm) placed on a 50 mL tube.
    ▲ Note: Moisten MACS SmartStrainer with buffer before use.
    ▲ Note: When upscaling the reagent volume and total volumes, increase also the number of MACS SmartStrainers (70 μm). One MACS SmartStrainer (70 μm) can be used for one adult mouse brain.
    ▲ Note: Dissociated tissue can be removed from the closed C Tube by pipetting through the septum-sealed opening in the center of the cap of the C Tube. Use ART® 1000 REACH™ 1000 μL pipette tips.
    ▲ Note: Cells with a diameter >70 μm may be lost. To obtain these cells within the flow through, use a cell strainer with an appropriate mesh size.
11. Apply 10 mL of cold (4 °C) D-PBS onto the MACS SmartStrainer (70 μm).
12. Discard MACS SmartStrainer (70 μm) and centrifuge cell suspension at 300×g for 10 minutes at 4 °C. Aspirate supernatant completely.
13. Proceed to "Debris and red blood cell removal".
    

Notes:

• Volumes given below are for the cell suspension from up to two adult mouse brains or up to 1 g of rat brain as starting material. When working with higher tissue quantities, scale up all reagent volumes accordingly.
• Cell suspensions from either two adult mouse brains or up to 1 g of rat brain are the maximum that can be processed in one 15 mL reagent tube.
• Always use pre-cooled buffers and solutions (4 °C).

▲ Note: In case of very small amount of tissue (< 100 mg) cell debris removal can be performed in a 5 mL reagent tube using 450 µL of Debris Removal Solution, 1550 µL of D-PBS for resuspension of the cell pellet, and 2000 µL D-PBS for overlay. 

1. Resuspend cell pellet carefully with the appropriate volume of cold D-PBS according to the table above and transfer cell suspension to a 15 mL tube. Do not vortex.
2. Add appropriate volume of cold Debris Removal Solution.
3. Mix well.
4. Overlay very gently with 4 mL of cold D-PBS.
   ▲ Note: Pipette very slowly to ensure that the D-PBS phase overlays the cell suspension and phases are not mixed.
5. Centrifuge at 4 °C and 3000×g for 10 minutes with full acceleration and full brake.
   ▲ Note: If centrifuges give suboptimal centrifugation, the acceleration and brake can be reduced.
6. Three phases are formed. Aspirate the two top phases completely and discard them.
7. Fill up with cold D-PBS to a final volume of 15 mL.
8. Gently invert the tube three times. Do not vortex.
9. Centrifuge at 4 °C and 1000×g for 10 minutes with full acceleration and full brake. Aspirate supernatant completely.
10. Resuspend cell pellet from up to two adult mouse brains carefully in 1 mL of cold 1× Red Blood Cell Removal Solution. Do not vortex.
11. Incubate for 10 minutes in the refrigerator (2−8 °C).
12. Add 10 mL of cold PB buffer.
13. Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.
14. Proceed to "Isolation of mouse microglia" or "Isolation of rat microglia".
    

Notes:

• Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
• Volumes for magnetic labeling given below are for up to 1×10⁷ total cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).
• For optimal performance it is important to obtain a single-cell suspension before magnetic labeling. Pass cells through 70 µm nylon mesh (MACS®  SmartStrainer (70 µm), # 130-098-462) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
• The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice may require increased incubation times.

1. Resuspend cell pellet carefully in 90 µL of cold PB buffer per 1×10⁷ total cells by pipetting slowly up and down. Do not vortex.
2. Add 10 µL of CD11b (Microglia) MicroBeads, human and mouse.
3. Mix well and incubate for 15 minutes in the dark in the refrigerator (2–8 °C)
4. Wash cells by adding 1 mL of cold PB buffer per 1×10⁷ cells and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
5. Resuspend up to 1×10⁷ cells in 500 µL of PB buffer.
   ▲Note: For higher cell numbers, scale up buffer volume accordingly.
6. (Optional) Take 20 µL for later flow cytometry analysis (original fraction).
7. Proceed to "Magnetic separation".
   

Magnetic separation with MS or LS Columns

• Choose an MS or LS Column and an appropriate MACS Separator. For details refer to "Materials".
• Always wait until the column reservoir is empty before proceeding to the next step.

1. Place column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
   
2. (Optional) Place Pre-Separation Filter (70 µm) on top of the column to remove clumps which may clog the column.
   ▲Note: Moisten Pre-Separation Filter with buffer before use.
3. Prepare column by rinsing with the appropriate amount of PB buffer:
   MS: 500 µL
   LS: 3 mL
4. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.
5. Wash column with the appropriate amount of buffer. Collect unlabeled cells that pass through and combine with the flow-through from step 4. This is the non-target cell fraction.
   MS: 3×500 µL
   LS: 3×3 mL
   ▲Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.
   
6. Remove column from the separator and place it on a suitable collection tube.
7. Pipette the appropriate amount of buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. This is the target cell fraction (positive fraction).
   MS: 1 mL
   LS: 5 mL
8. To increase the purity of microglia cells, it is recommended to enrich the positive fraction over a second MS or LS Column. Repeat the magnetic separation procedure as described in steps 1 to 7 using a new column.
9. Proceed to"Flow cytometry analysis of mouse microglia".
   

Magnetic separation with the autoMACS® Pro Separator

Notes:

• Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator.
• Use PBS buffer. Buffers used for operating the autoMACS Pro Separator should have a temperature of ≥10 °C.

1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C.
3. For a standard separation choose the following program:
   Positive selection of mouse microglia cells: Possel
   Collect positive fraction in row C of the tube rack. This fraction represents the target cells.
4. (Optional) Collect negative fraction in row B of the tube rack. This fraction represents the non-target cells.
5. Proceed to "Flow cytometry analysis of mouse microglia".

Notes:

• Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling.
• Volumes for magnetic labeling given below are for up to 1×10⁷ total cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 1×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).
• For optimal performance it is important to obtain a single-cell suspension before magnetic labeling. Pass cells through 70 µm nylon mesh (MACS®  SmartStrainer (70 µm), # 130-098-462) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
• The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice may require increased incubation times.

1. Resuspend cell pellet carefully in 80 µL of cold PB buffer per 1×10⁷ total cells by pipetting slowly up and down. Do not vortex.
2. Add 20 µL of CD11b (Microglia) MicroBeads, rat.
3. Mix well. Do not vortex. Incubate for 15 minutes in the dark in the refrigerator (2–8 °C).
4. Wash cells by adding 1 mL of cold PB buffer per 1×10⁷ cells and centrifuge at 300×g for 5 minutes. Aspirate supernatant completely.
5. Resuspend up to 1×10⁷ cells in 500 µL of PB buffer.
   ▲Note: For higher cell numbers, scale up buffer volume accordingly.
6. (Optional) Take 20 µL for later flow cytometry analysis (original fraction).
7. Proceed to "Magnetic separation".
   

Magnetic separation with MS or LS Columns

• Choose an MS or LS Column and an appropriate MACS Separator. For details refer to "Materials".
• Always wait until the column reservoir is empty before proceeding to the next step.

1. Place column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
   
2. (Optional) Place Pre-Separation Filter (70 µm) on top of the column to remove clumps which may clog the column.
   ▲Note: Moisten Pre-Separation Filter with buffer before use.
3. Prepare column by rinsing with the appropriate amount of PB buffer:
   MS: 500 µL
   LS: 3 mL.
4. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.
5. Wash column with the appropriate amount of buffer. Collect unlabeled cells that pass through and combine with the flow-through from step 4. This is the non-target cell fraction.
   MS: 3×500 µL
   LS: 3×3 mL.
   ▲Note: Perform washing steps by adding buffer aliquots only when the column reservoir is empty.
   
6. Remove column from the separator and place it on a suitable collection tube.
7. Pipette the appropriate amount of buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. This is the target cell fraction (positive fraction).
   MS: 1 mL
   LS: 5 mL.
8. To increase the purity of microglia cells, it is recommended to enrich the positive fraction over a second MS or LS Column. Repeat the magnetic separation procedure as described in steps 1 to 7  using a new column.
9. Proceed to "Flow cytometry analysis of rat microglia".
   

Magnetic separation with the autoMACS® Pro Separator

Notes:

• Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator.
• Use PBS buffer. Buffers used for operating the autoMACS Pro Separator should have a temperature of ≥10 °C.

1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place sample tube in row A of the tube rack and the fraction collection tubes in rows B and C.
3. For a standard separation choose the following program:
   Positive selection of rat microglia cells: Posseld2
   Collect positive fraction in row C of the tube rack. This fraction represents the target cells.
4. (Optional) Collect negative fraction in row B of the tube rack. This fraction represents the non-target cells.
5. Proceed to "Flow cytometry analysis of rat microglia".

Notes:

• The recommended antibody dilution for labeling of cells is 1:10 for up to 1×10⁷ cells/100 μL of buffer.
• Volumes given below are for up to 1×10⁷ nucleated cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×10⁷ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).

1. (Optional) For analysis take 100 µL of positive and negative fraction. Include the 20 µL of the original fraction.
2. Resuspend up to 1×10⁷ nucleated cells per 100 μL of buffer.
3. Add 10 μL of CD11b-PE or CD11b-APC.
4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2-8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specifc cell labeling. Working on ice requires increased incubation times
5. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.

Notes:

• The recommended antibody dilution for labeling of cells and subsequent analysis by flow cytometry is 1:10 for up to 1×10⁶ cells/50 μL of buffer.
• Volumes given below are for up to 1×10⁶ nucleated cells. When working with fewer than 1×10⁶ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×10⁶ nucleated cells, use twice the volume of all indicated reagent volumes and total volumes). 
  

1. (Optional) For analysis take 100 µL of positive and negative fraction. Include the 20 µL of the original fraction.
2. Resuspend up to 1×10⁶ nucleated cells per 45 μL of buffer.
3. Add 5 μL of CD11b/c-PE or CD11b/c-APC.
4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2–8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead t nonspecific cell labeling. Working on ice requires increased incubation times.
5. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.
   

1. Plate 1×10⁵ cells in 50 µL of prepared medium as a drop in the middle of each well of a 24-well plate which has been coated overnight (see “Things to prepare in advance of tissue dissociation and cell culture”).
2. Let the cells settle down for 30 minutes at 37 °C in the incubator.
3. Carefully add 450 µL of prepared medium to each well.
4. Maintain the culture by replacement of 50% of prepared medium every other day.
   ▲Note: Cells loosely attach after the first days of cultivation. Therefore, a cultivation period of approximately 1 week is recommended. 
   

1. Wash cells 3× with PBS.
2. Fix cells with 2% PFA for 10 minutes at room temperature.
3. Wash cells 3× with PBS.
   ▲Note: When working with CD68 antibodies, add 0.2% TRITON™ X-100 in PBS, incubate for 10 minutes at room temperature, and wash cells 3× with autoMACS Running Buffer.
   ▲Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
4. Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
5. Discard staining buffer.
6. Add primary antibody in staining buffer to the cells with a final concentration of 5–10 μg/mL and incubate at room temperature in the dark for 10 minutes.
   When working with CD68 antibodies: Incubate at 2–8 °C overnight in the dark.
7. Wash cells 3× with autoMACS Running Buffer.
8. Add a corresponding secondary antibody in staining buffer to the cells.
   When working with CD11b antibodies: Incubate at room temperature in the dark for 10 minutes.
   When working with CD68 antibodies: Incubate at room temperature in the dark for 1 hour.
9. Wash cells 3× with autoMACS Running Buffer.
   ▲Note: For co-staining with additional antibodies repeat steps 6–9.
10. Store cells in autoMACS Running Buffer.
11. Cells are now ready for immunofluorescence microscopy.
    ▲Note: Samples can be stored at 2–8 °C in the dark for up to one week
    ▲Note: When working with cells cultured on coverslips, the coverslips need to be mounted onto slides before imaging.
    

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