MACS Tissue Dissociation Kits搭配 gentlemacs系列组织处理器,能够温和、高效地离神经组织:

 - 全自动、高效的组织解离；

 - 结果可靠,不依赖于操作者；

 - 高得率、高活性的单细胞悬液；

 - 过程温和,保护细胞和表面抗原表位的完 整性；

 - 最理想的样品制备方法,完美兼容下游实验应用，包括分选出多种类型的神经细胞。

 图1：带加热模块gentleMACS Octo全自动组织处理器——多功能、简便可靠的台式组织处理器。

配合 gentlemacs组织处理器,利用神经组织解离试剂盒可以自动化解离神经组织(≤P7),结合解离和机械解离,获得高质量的单细胞悬液。利用试剂盒中高效的酶解离,可以获得高得率、高活性的单细胞,并保护细胞表面抗原表位的完整性。该试剂盒适用于整个大脑或组织切片的解离。

图2:使用神经组织解离试剂盒和 gentlemacsf组织处理器进行标准化的组织解离。 Neural Tissue Dissociation Kit(P)搭配带加热模块gentleMACS Dissociator全自动组织处理器将小鼠的経个大脑组织(P4)进行解离。获得的细胞使用PI染色并进行流式细胞分析。结果表明,获得的单细胞悬液细胞碎片非常少A,而活细胞比例极高(B)。

实验成功与否在很大程度上依赖于起始样品制备的质量。在处理比较脆弱的组织(如成年脑)的时候,这点尤为重要。当处理的神经组织是来源于>P7龄的动物时,大量的细胞碎片和红细胞往往影响下游实验应用。 Adult Brain Dissociation Kit, mouse and rat,含有去除细胞碎片和红细胞的相关试剂,可以轻松解决这一问题,获得高纯度、高活性的神经细胞,兼容下游实验,如细胞分选流式细胞分析、单细胞测序或细胞移植。使用 Adult Brain Dissociation Kit,搭配 gentleMACS系列组织处理器,去除碎片和红细胞 - 做磁性细胞分选时,抗体结合效率更高； - 提高抗体的利用率,更高效地进行免疫荧光染色； - 蛋白免疫印迹和流式细胞分析等实验更高质量的细胞培养。

图3:使用 Adult Brain Dissociation Kit, mouse and rat,获得高得率、高活性、高纯度的神经细跑。成年鼠大脑经解离得到的单细胞悬液含有大量的细胞碎片和红细胞(A),这会妨碍下游的细跑分选、培养和分析等实验。 Adult Brain Dissociation Kit. mouse and rat优化了试剂和组织解离方法,获得的单细胞悬液中仅含有少量的细胞碎片和红细胞(B)。

您会同时处理多个样本吗,比如24个样本?您是否经历过因为大量髓鞘碎片的存在而妨碍下游实验的情况?Myelin Removal Beads(MRBD)是专为从神经细胞悬液中去除髓鞘碎片而设计的(已测试过小鼠、大鼠、人和绵羊的样品),与 MULTIMACS Cell245 eparator Plust仪器完全兼容(图5),可以同时平行处理多达24个样品。

图4：使用Myelin Removal Beads ll有效地从单细胞悬液中去除髓鞘碎片。使用Neural Tissue Dissociation Kit(P)解离试剂盒解离出生P22龄的小鼠大脑，获得的单细胞悬液用Myelin Removal Beads ll去除髓鞘碎片，然后用流式分析髓鞘去除前(A)和髓鞘去除后(B)的细胞。

MACS磁分选技术是经过广泛验证的、可以用于基础和临床研究的最佳细胞分选技术，有超过20000篇文献引用。使用MACS磁珠进行细胞分选，您将体验到： - 高效：仅1小时内分选获得神经细胞； - 可靠：获得的细胞活力高、纯度高； - 灵活：使用美天旎间标磁珠，可以分选几乎任意物种的任意类型细胞，间标磁珠如：anti-lgG/lgM、anti-Biotin/FITC/P E/APC磁珠等。
手动细胞分选：结合MACS分选柱和分选器，利用MACS磁珠可以进行灵活、高效的神经细胞分选。

图5：(A)使用OctoMACS分选器、MS分选柱和MACS分选架进行快速手动细胞分选。(B)使用MultiMACS Ce1124 Separator Plus和autoMACS Pro Separator进行自动化磁性细胞分选。
多个细胞样品的自动化分选MACS磁珠可以与autoMACS  Pro磁性细胞分选仪和MultiMACS^TM Ce1124分选仪完全兼容，实现自动化的细胞分选，并能同时处理多个样品，方便、高效。

脑组织解离试剂

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 or brain tissue from adult rats or distinct brain regions, determine the weight and 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.

• 对于随后的细胞分离和培养，建议分离至少800 mg成年小鼠或大鼠脑组织。

• 下面给出的体积是一个成年小鼠大脑(最大500mg）在1980μL酶混合液中。 当操作少于500 mg以下时，使用与指示相同的体积。 当处理来自成年大鼠或不同脑区的较高数量组织或脑组织时，确定重量，并相应地扩大所有试剂体积和总体积。 每C管最多可处理500 mg脑组织。

准备小鼠和大鼠成年脑组织解离试剂盒

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.

准备木瓜蛋白酶(P)备用。 准备适当体积的分装液避免反复冻融。 在-20°C存储分装管。 此溶液稳定期为6个月。用1 mL缓冲液A将冻干粉重悬在标有酶A的小瓶中。不要涡旋。 该溶液应该被分装并存储在-20°C，避免反复冻融。

2. Prepare enzyme mix 1 and enzyme mix 2 according to the table below.

根据下表准备酶混合液1和酶混合液2：

Enzyme mix 1

Enzyme mix 2

Enzyme P

50 µL

Buffer Z

1900 µL

Buffer Y

20 µL

Enzyme A

10 µL

准备1x红细胞去除试剂

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.

用双蒸水(ddH₂O)以1:10稀释红细胞去除液（10×），例如用0.9mL冷ddH₂O稀释0.1mL的冷的红细胞去除液。

▲注意：不要用去离子水稀释。

2. Store the prepared 1× Red Blood Cell Removal Solution at 2–8 °C. Discard unused solution at the end of the day.

在2-8℃存储1 x红细胞裂解液。在一天结束时丢弃未使用的试剂。

准备细胞培养皿

1. Prepare the following medium: MACS® Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine, 10 ng/mL Human PDGF-AA, and 10 ng/mL Human FGF-2.

制备以下细胞培养基：MACS Neuro Medium+2%MACS NeuroBrew®-21+1%青霉素/链霉素+0.5mM-谷氨酰胺。

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. Let the culture dish dry under sterile conditions.

用0.01%聚-L-赖氨酸包被24孔培养皿，37℃过夜。 第二天用ddH₂O洗三次。

01.解离脑组织

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".

02.去除碎片和红细胞

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.

• The cell suspension from two adult mouse brains or up to 1 g of rat brain is the maximum that can be processed in one 15 mL reagent tube.

• Always use pre-cooled buffers and solutions (4 °C).

Debris Removal Solution

D-PBS

Overlay (D-PBS)

1 brain(400-500 mg)

900 µL

3100 µL

4 mL

2 brains(800-1000 mg)

1800 µL

6200 µL

4 mL

▲ 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 D-PBS/BSA buffer.

13.Centrifuge at 4 °C and 300×g for 10 minutes. Aspirate supernatant completely.

14.Proceed to "Isolation of oligodendrocytes". 

03.分离小鼠小胶质细胞

注意：

• 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.

• 快速操作，细胞保持在冷的状态，使用预冷溶液。 这将防止抗体盖在细胞表面和非特异性细胞标记。

• 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).

• 下面给出的磁性标记的体积最多为1×10⁷总细胞。 当使用少于1×10⁷细胞数时，使用与指示相同的体积。 当使用较高的细胞数时，相应地扩大所有试剂体积和总体积（例如，对于2×10⁷的总细胞，使用所有指示试剂体积和总体积的两倍）。

• 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 D-PBS/BSA buffer before use.

• 为了获得最佳的性能，在磁标记之前获得单细胞悬浮液是很重要的。 通过70µm尼龙网(MACS®  SmartStrainer（70µm），#130-098-462)消除可能堵塞柱的细胞团块。 使用前用缓冲液润洗过滤器。

• 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.

• 推荐的孵育温度为2-8°C。 较高的温度和/或较长的孵育时间可能导致非特异性细胞标记。 在冰上工作可能需要增加孵化时间。

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.

小心用 90 µL PB 缓冲液重悬 1×10⁷总细胞，不要涡旋。

2. Add 10 µL of CD11b (Microglia) MicroBeads, human and mouse.

加入10 µL抗CD11b小胶质细胞分选磁珠，小鼠和人。

3. Mix well and incubate for 15 minutes in the dark in the refrigerator (2–8 °C)

混匀，2-8℃，避光孵育15分钟。

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.

加入1 mLPB缓冲液，300xg离心5分钟洗涤细胞。完全弃去上清液。

5. Resuspend up to 1×10⁷ cells in 500 µL of PB buffer.

用 500 µL PB缓冲液重悬 1×10⁷总细胞.

▲Note: For higher cell numbers, scale up buffer volume accordingly.

▲注意：对于更多数量的细胞，相应增加缓冲液体积。

6. (Optional) Take 20 µL for later flow cytometry analysis (original fraction).

（可选）吸取20µL用于后期的流式分析（初始部分）

7. Proceed to "Magnetic separation".

进行磁珠分选。

Magnetic separation with MS or LS Columns

Notes:

• 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".

04.分离大鼠小胶质细胞

注意：

• 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.

• 快速操作，细胞保持在冷的状态，使用预冷溶液。 这将防止抗体盖在细胞表面和非特异性细胞标记。

• 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).

• 下面给出的磁性标记的体积最多为1×10⁷总细胞。 当使用少于1×10⁷细胞数时，使用与指示相同的体积。 当使用较高的细胞数时，相应地扩大所有试剂体积和总体积（例如，对于2×10⁷的总细胞，使用所有指示试剂体积和总体积的两倍）。

• 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 D-PBS/BSA buffer before use.

• 为了获得最佳的性能，在磁标记之前获得单细胞悬浮液是很重要的。 通过70µm尼龙网(MACS®  SmartStrainer（70µm），#130-098-462)消除可能堵塞柱的细胞团块。 使用前用缓冲液润洗过滤器。

• 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.

• 推荐的孵育温度为2-8°C。 较高的温度和/或较长的孵育时间可能导致非特异性细胞标记。 在冰上工作可能需要增加孵化时间。

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.

小心用 90 µL PB 缓冲液重悬 1×10⁷总细胞，不要涡旋。

2. Add 10 µL of CD11b (Microglia) MicroBeads, human and mouse.

加入10 µL抗CD11b小胶质细胞分选磁珠，小鼠和人。

3. Mix well and incubate for 15 minutes in the dark in the refrigerator (2–8 °C)

混匀，2-8℃，避光孵育15分钟。

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.

加入1 mLPB缓冲液，300xg离心5分钟洗涤细胞。完全弃去上清液。

5. Resuspend up to 1×10⁷ cells in 500 µL of PB buffer.

用 500 µL PB缓冲液重悬 1×10⁷总细胞.

▲Note: For higher cell numbers, scale up buffer volume accordingly.

▲注意：对于更多数量的细胞，相应增加缓冲液体积。

6. (Optional) Take 20 µL for later flow cytometry analysis (original fraction).

（可选）吸取20µL用于后期的流式分析（初始部分）

7. Proceed to "Magnetic separation".

进行磁珠分选。

Magnetic separation with MS or LS Columns

Notes:

• 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".

04.小胶质细胞流式分析

Notes:

• The recommended antibody dilution for labeling of cells is 1:11 for up to 1×10⁷ cells/100 μL of D-PBS/BSA 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. You can also analyze 20 µL of the original fraction.

2.Resuspend up to 1×10⁷ nucleated cells per 100 μL of D-PBS/BSA buffer.

3.Add 10 μL of Anti-O4-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-specific cell labeling. Working on ice requires increased incubation times.

5.Wash cells by adding 1 mL of D-PBS/BSA buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.

6.Resuspend cell pellet in a suitable amount of D-PBS/BSA buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10. 

05.少突胶质细胞培养

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.

06.培养细胞免疫化学染色

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

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: 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 Anti-O4 pure 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.

7.Wash cells 3× with autoMACS Running Buffer.

8.Add a corresponding secondary antibody (anti-mouse IgM) in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.

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.

试剂、耗材

操作步骤

- Neural Tissue Dissociation Kit (T) (# 130-093-231) or 

Neural Tissue Dissociation Kit (P) (# 130-092-628)

- Hanks´ Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55021C)

- HBSS with Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55037C)

- (Optional) β-mercaptoethanol, 50 mM

- 50 mL tubes

- MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tube

- MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C

- gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)

- C Tubes (# 130-093-237, # 130-096-334)

- (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

-  (Optional) Myelin Removal Beads II, human, mouse, rat (# 130-096-433, # 130-096-733)

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.

Neural Tissue Dissociation Kit (T) (# 130-093-231)

Neural Tissue Dissociation Kit (P) (# 130-092-628)

02.分离星形胶质细胞

试剂、耗材

操作步骤

- Anti-ACSA-2 MicroBead Kit, mouse (# 130-097-678) or Anti-ACSA-2 MicroBead Kit, mouse - small (# 130-097-679)

- Pre-Separation Filters (70 μm) (# 130-095-823)

- PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with PBS. 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).

- MACS Columns and MACS Separators: GLAST+ cells can be enriched by using MS or LS Columns. Positive selection can also be performed by using the autoMACS® Pro Separator or the MultiMACS™ Cell24 Separator. 

Isolate the ACSA-2-positive astrocytes from the single-cell suspension using the Anti-ACSA-2 MicroBead Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

 Anti-ACSA-2 MicroBead Kit, mouse (# 130-097-678)

03.流式分析星形胶质细胞

试剂、耗材

操作步骤

• Fluorochrome-conjugated Anti-ACSA-2 antibodies for flow cytometry analysis, e.g., Anti-ACSA-2-APC (# 130-102-315). Learn more about our antibodies and dyes.

• Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cell

• MACSQuant® Analyzer 10 (# 130-096-343)

Notes:

• The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of PB 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.Use 100 μL of the ACSA-2⁺ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.

2.Resuspend up to 1×10⁶ nucleated cells per 45 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
▲ Note: Always use freshly prepared buffer.

3.Add 5 μL of Anti-ACSA-2-APC, mouse antibodies.

4.Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.

5.Wash cells by adding 1 mL of PB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.

6.Resuspend cell pellet in a suitable amount of PB buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.

04.细胞培养

试剂、耗材

操作步骤

- Double-distilled water (ddH₂O)

- Imaging Plate CG 1.5 (24 well) (# 130-098-263)

- Poly-L-lysine (0.01%)

- Penicillin/streptomycin

- MACS Neuro Medium (# 130-093-570) 

- MACS NeuroBrew®-21 (# 130-093-566)

- L-glutamine

试剂准备：

- Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

- repare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

步骤：

1. Plate 5×10⁴ cells in 50 μL of pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of cell isolation 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 replacing 50% of medium every other day.

05.细胞免疫染色

试剂、耗材

操作步骤

- Anti-GLAST (ACSA-1) pure, human, mouse, rat (# 130-095-822) and anti-rat-IgG2b secondary antibody or Anti-ACSA-2 pure, mouse (# 130-099-138) and anti-mouse-IgG2a secondary antibody

- Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

- Phosphate-buffered saline (PBS)

- FcR Blocking Reagent, mouse (# 130-092-575)

- autoMACS Running Buffer (# 130-091-221)

- 2% paraformaldehyde (PFA) for fixation

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

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: 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.

5. Add primary antibody in staining buffer to the cells with a final concentration of 1–5 μg/mL and incubate at room temperature in the dark for 10 minutes.

6. Wash cells 3× with autoMACS Running Buffer.

7. Add a corresponding secondary antibody in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.

8. Wash cells 3× with autoMACS Running Buffer.
▲Note: For co-staining with additional antibodies repeat steps 6–9.

9. Store cells in autoMACS Running Buffer.

10. 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.

P3 whole mouse brains were dissociated using the Neural Tissue Dissociation Kit (T) and astrocytes were isolated from the single-cell suspension using the Anti-GLAST MicroBead Kit. Astrocytes were cultured in MACS Neuro Medium, MACS NeuroBrew-21, 1% P/S, and 0.5 mM L-glutamine on PLL-coated glass coverslips. After 3 days (A) and 6 days (B), cells were fixed and stained with astrocyte-specific antibodies Anti-GLAST (green) and Anti-GFAP (red).

试剂、耗材

操作步骤

- Neural Tissue Dissociation Kit (T) (# 130-093-231)

- Hanks´ Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55021C)

- HBSS with Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55037C)

- (Optional) β-mercaptoethanol, 50 mM

- 50 mL tubes

- MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tube

- MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C

- gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)

- C Tubes (# 130-093-237, # 130-096-334)

- (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

-  (Optional) Myelin Removal Beads II, human, mouse, rat (# 130-096-433, # 130-096-733)

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.

Neural Tissue Dissociation Kit (T) (# 130-093-231)

02.分离星形胶质细胞

试剂、耗材

操作步骤

- Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat (# 130-095-826, # 130-095-825)

- Pre-Separation Filters (70 μm) (# 130-095-823)

- PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with PBS. 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).

- MACS Columns and MACS Separators: GLAST+ cells can be enriched by using MS or LS Columns. Positive selection can also be performed by using the autoMACS® Pro Separator or the MultiMACS™ Cell24 Separator. 

Isolate the GLAST-positive astrocytes from the single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit. Follow the protocol of the kit data sheet.

Download data sheet

Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat

Effective isolation of GLAST⁺ cells from mouse brain tissue. 

Mouse brain tissue (P7) was dissociated using the Neural Tissue Dissociation Kit (T), the gentleMACS™ Dissociator, and FcR Blocking Reagent, mouse. Astrocytes were isolated from single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit, a MiniMACS™ Separator and an MS Column. Cells were fluorescently stained with Anti-GLAST (ACSA-1)-APC and analyzed by flow cytometry on the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

03.流式分析星形胶质细胞

试剂、耗材

操作步骤

- FcR Blocking Reagent, mouse (#130-092-575) to avoid Fc receptor-mediated antibody labeling

- Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., Anti-GLAST (ACSA‑1)-PE
(# 130‑095‑821) or Anti-GLAST (ACSA-1)-APC (# 130‑095‑814). Learn more about our antibodies and dyes.

- Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cell

- MACSQuant® Analyzer 10 (# 130-096-343)

Notes:

• The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of PB 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. Use 100 μL of the GLAST+ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.

2. Resuspend up to 1×10⁶ nucleated cells per 45 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
▲ Note: Always use freshly prepared buffer.

3. Add 5 μL of Anti-GLAST (ACSA-1)-APC, mouse antibodies.

4. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.

5. Wash cells by adding 1 mL of PB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.

6. Resuspend cell pellet in a suitable amount of PB buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.

04.细胞培养

试剂、耗材

操作步骤

- Double-distilled water (ddH₂O)

- Imaging Plate CG 1.5 (24 well) (# 130-098-263)

- Poly-L-lysine (0.01%)

- Penicillin/streptomycin

- Fetal bovine serum (FBS)

- MACS Neuro Medium (# 130-093-570) 

- MACS NeuroBrew®-21 (# 130-093-566)

- L-glutamine

试剂准备：

- Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

- repare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

步骤：

1. Plate 5×10⁴ cells in 50 μL of pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of cell isolation 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 replacing 50% of medium every other day.

05.细胞免疫染色

试剂、耗材

操作步骤

- Anti-GLAST (ACSA-1) pure, human, mouse, rat (# 130-095-822) and anti-rat-IgG2b secondary antibody or Anti-ACSA-2 pure, mouse (# 130-099-138) and anti-mouse-IgG2a secondary antibody

- Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

- Phosphate-buffered saline (PBS)

- FcR Blocking Reagent, mouse (# 130-092-575)

- autoMACS Running Buffer (# 130-091-221)

- 2% paraformaldehyde (PFA) for fixation

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

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: 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.

5. Add primary antibody in staining buffer to the cells with a final concentration of 1–5 μg/mL and incubate at room temperature in the dark for 10 minutes.

6. Wash cells 3× with autoMACS Running Buffer.

7. Add a corresponding secondary antibody in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.

8. Wash cells 3× with autoMACS Running Buffer.
▲Note: For co-staining with additional antibodies repeat steps 6–9.

9. Store cells in autoMACS Running Buffer.

10. 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.

P3 whole mouse brains were dissociated using the Neural Tissue Dissociation Kit (T) and astrocytes were isolated from the single-cell suspension using the Anti-GLAST MicroBead Kit. Astrocytes were cultured in MACS Neuro Medium, MACS NeuroBrew-21, 1% P/S, and 0.5 mM L-glutamine on PLL-coated glass coverslips. After 3 days (A) and 6 days (B), cells were fixed and stained with astrocyte-specific antibodies Anti-GLAST (green) and Anti-GFAP (red).

试剂、耗材

操作步骤

- Neural Tissue Dissociation Kit (T) (# 130-093-231) or Neural Tissue Dissociation Kit (P) (# 130-092-628)

- Hanks´ Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55021C)

- HBSS with Ca²⁺ and Mg²⁺ (Sigma-Aldrich # 55037C)

- 50 mL tubes

- (Optional) Beta-mercaptoethanol, 50 mM

- MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tube

- MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C

- gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)

- C Tubes (# 130-093-237, # 130-096-334)

- (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.

Use the Neural Tissue Dissociation Kit (T) or the Neural Tissue Dissociation Kit (P) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.

02.少突胶质细胞分离及流式分析

试剂、耗材

操作步骤

 - CD140a (PDGFRα) MicroBead Kit, mouse (#130-101-502; small size 130-101-547)

 - Pre-Separation Filters (70 µm) (#130-095-823)

 - CD140a流式抗体
▲Note: The use of CD140a antibodies, clone APA5, is not recommended for analysis of cells that are labeled with CD140a (PDGFRα) MicroBeads.

 - Propidium Iodide Solution (#00-6990-50) or 7-AAD（#00-6993-50） for flow cytometric exclusion of dead cells.

 - Immunosep buffer(#604025)

 - MACS Columns and MACS Separators: CD140a (PDGFRα)+ cells can be enriched using LS or MS Columns. Positive selection can also be performed using the autoMACS Pro Separator.

Isolate oligodendrocyte precursor cells using the CD140a (PDGFRα) MicroBead Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

CD140a (PDGFRα) MicroBead Kit, mouse

To stain and analyze the isolated oligodendrocyte precursor cells by flow cytometry, follow the protocol on cell surface flow cytometry staining.

03.细胞培养

试剂、耗材

操作步骤

- Double-distilled water (ddH₂O)

- Imaging Plate CG 1.5 (24 well) (# 130-098-263)

- L-glutamine

- Poly-L-lysine (0.01%)

- Penicillin/streptomycin

- MACS Neuro Medium (# 130-093-570)

- MACS NeuroBrew®-21 (# 130-093-566)

- Penicillin/streptomycin

- Human PDGF-AA (# AF-100-13A-2)

- Human FGF-2 (# 100-18C-10) 

试剂准备：

- 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, 10 ng/mL Human PDGF-AA, and 10 ng/mL Human FGF-2.

步骤：

1. Plate 5×10⁴ cells in 50 µL of prepared medium as a drop in the middle of each well of a 24-well plate that has been coated overnight .

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 replacing 50% of prepared medium every other day.
▲Note: If oligodendrocytes are cultivated for longer periods, e.g., 2 weeks, we recommend increasing FGF-2 and PDGF-AA concentrations to 20 ng/mL instead of 10 ng/mL.

05.细胞免疫染色

试剂、耗材

操作步骤

- Anti-O4 pure, human, mouse, rat (# 130-115-810) and anti-mouse IgM secondary antibody

- Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

- Phosphate-buffered saline (PBS)

- autoMACS Running Buffer (# 130-091-221)

 -2% paraformaldehyde (PFA) for fixation

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.

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: 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 Anti-O4 pure 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.

7.Wash cells 3× with autoMACS Running Buffer.

8.Add a corresponding secondary antibody (anti-mouse IgM) in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.

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.