PBE Buffer (standard wash and dilution buffer)

Prepare a solution of PBS, pH 7.2, 2mM EDTA and 0.5% BSA by diluting MACS^® BSA Stock Solution 1:20 with autoMACS^® Rinsing Solution.

Notes:

• For details on the use of the gentleMACS™ Dissociators, refer to the gentleMACS Dissociator user manuals.
• For cell culture experiments subsequent to tissue dissociation, all steps should be performed under sterile conditions.
• The weight of one mouse spleen amounts to 80–120 mg (female BALB/c mouse, 6–7 weeks old).

1. Transfer mouse spleen into the gentleMACS C Tube containing the following amount of PBE buffer:

   1–2 mouse spleens: 3 mL
   3–4 mouse spleens: 6 mL
   5–6 mouse spleens: 9 mL
   
   
2. Close C Tube tightly and attach it upside down onto the sleeve of the gentleMACS Dissociator. 
   ▲ Note: Close C Tube tightly beyond the first resistance. 
   ▲ Note: Ensure that the sample material is located in the area of the rotor/stator.
3.  Choose and run one of the following gentleMACS Programs:
   1–2 mouse spleens: m_spleen_01
   3–6 mouse spleens: m_spleen_04
4. After termination of the program, detach C Tube from the gentleMACS Dissociator.
5. (Optional) Perform a short centrifugation step to collect the sample material at the bottom of the tube.
6. Resuspend sample and apply the cell suspension to a Pre-Separation Filter, 30 μm, placed on a 15 mL tube (1–2 mouse spleens per C Tube) or to an appropriate cell strainer placed on a 50 mL tube (3–6 mouse spleens per C Tube).
   ▲ 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.
7. Wash Pre-Separation Filter with 5 mL of PBE buffer.
8. Discard Pre-Separation Filter and centrifuge cell suspension at 300×g for 10 minutes at room temperature. Aspirate supernatant completely.
9. Resuspend cells in PBE buffer to the required volume for further applications. For example, resuspend cells in 10 mL buffer for magnetic labeling.
   ▲Note: Process cells immediately.

1. Prepare cells and determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in 400 µL of PBE buffer per 1×10⁸ total cells.
4. Add 100 µL of Biotin-Antibody Cocktail per 1×10⁸ total cells.
5. Mix well and incubate for 5 minutes in the refrigerator (2−8 °C).
6. Add 200 µL of PBE buffer per 1×10⁸ total cells.
7. Add 200 µL of Anti-Biotin MicroBeads per 1×10⁸ total cells.
8. Add 100 µL of CD44 MicroBeads per 1×10⁸ total cells.
9. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C).
10. (Optional) For highest recovery, wash cells by adding 1–2 mL of PBE buffer per 1×10⁸ total cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely. Resuspend up to 1×10⁸ cells in 500 µL of PBE Buffer.
11. Proceed to "Magnetic separation".

Notes:

• Always wait until the column reservoir is empty before proceeding to the next step.
• Choose an LS Column and a suitable MACS® Separator.

1. Place LS Column in the magnetic field of a suitable MACS Separator. For details refer to the respective MACS Column data sheet.
2. Prepare column by rinsing with 3 mL of PBE buffer.
3. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells, representing the enriched naïve CD4⁺ T cells.
4. Wash column with 3 mL of PBE buffer. Collect unlabeled cells that pass through, representing the enriched naïve CD4⁺ T cells, and combine with the effluent from step 3.
   ▲ Note: (Optional) If flow cytometry analysis of the freshly isolated naïve CD4⁺ T cells is desired, take an aliquot (up to 1×10⁶ cells) and proceed to "Flow cytometry analysis of freshly isolated naïve CD4⁺ T cells".
5. (Optional) Remove column from the separator and place it on a suitable collection tube. Pipette 5 mL of PBE buffer onto the column. Immediately flush out the magnetically labeled non-naive CD4⁺ T cells by firmly pushing the plunger into the column.

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. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend up to 1×10⁶ nucleated cells per 25 µL of PBE buffer.
4. Add 5 µL of each antibody:
   CD45-VioGreen™ (# 130-102-412)
   CD4-VioBlue® (# 130-102-456)
   CD62L-PE (# 130-102-543)
   CD3ε-APC-Vio®770 (# 130-102-306)
   CD44-FITC (# 130-102-511)
5. 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.
6. Wash cells by adding 1−2 mL of PBE buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Resuspend cell pellet in a suitable amount of PBE buffer (e.g. 500 µl) for analysis by flow cytometry or fluorescence microscopy.
   ▲ Note: Add propidium iodide to a final concentration of 1 µl per mL (e.g., 0.5 µl in 500 µl) to the cell suspension, if exclusion of dead cells via fluorescence is desired.

Supplemented T cell medium

TexMACS™  medium supplemented with FBS (final concentration 10%), 2-mercaptoethanol (final concentration 0.01 mM) and 100× penicillin/streptomycin stock solution (final concentration 1%).

Notes:

• Medium referred to as fully supplemented T cell medium (see "Polarization of naïve T cells") consists of supplemented T cell medium (TexMACS including 10% FBS, 0.01 nM 2-mercaptoethanol and 1% penicillin/streptomycin stock solution) with the addition of cytokines and antibodies from the CytoBoxes.
• Addition of penicillin/streptomycin to the T cell media is optional.

Loading of Anti-Biotin MACSiBead™ Particles

• Resuspend Anti-Biotin MACSiBead Particles thoroughly by vortexing before use, to obtain a homogenous suspension.
• Anti-Biotin MACSiBead Particles are supplied without preservative. Remove aliquots under aseptic conditions.
• It is recommended to load Anti-Biotin MACSiBead Particles in batches of 1×10⁸ Anti-Biotin MACSiBead Particles. Loaded Anti-Biotin MACSiBead Particles are stable for up to 4 months when stored at 2–8 °C.

1. Pipette 100 μL of CD3ε-Biotin and 100 μL CD28-Biotin into a sealable 2 mL tube and mix well.
   ▲ Note: This antibody combination, with a final antibody concentration of 10 μg antibody per 1 mL loaded Anti-Biotin MACSiBead Particles, is optimized for achieving maximum T cell activation.
2. Add 300 μL of buffer and mix well.
3. Resuspend Anti-Biotin MACSiBead Particles thoroughly by vortexing.
4. Remove 500 μL Anti-Biotin MACSiBead Particles (1×10⁸ Anti-Biotin MACSiBead Particles) and add to antibody mix.
   ▲ Note: Anti-Biotin MACSiBead Particles can be loaded in a flexible manner with biotinylated antibodies or ligands other than those supplied. If desired, add other biotinylated antibodies or ligands at appropriate concentrations and adjust with buffer to a total volume of 1 mL accordingly.
5. Incubate for 2 hours at 2–8 °C under constant, gentle rotation by using the MACSmix™ Tube Rotator at approximately 4 rpm (slowest permanent run program).
6. The loaded Anti-Biotin MACSiBead Particles (1×10⁸ Anti-Biotin MACSiBead Particles/mL) are now ready to use. Do not remove the loaded Anti-Biotin MACSiBead Particles from the antibody mix. Store at 2–8 °C for up to 4 months.

Calculation of cytokine concentration

In order to obtain maximal reproducibility for your T_H cell differentiation experiments, it is recommended to always dose recombinant cytokines at a defined unit dose in [U/mL]. To calculate the cell culture concentration in [ng/mL] corresponding to the concentration in [U/mL], apply the following formula:

Final culture concentration [ng/mL] = (60 U/mL)/(biological activity [U/mg]*) × (10⁶)

* Refer to the corresponding data sheet or CoA to obtain the biological activity.

Plate sizes for in vitro T cell polarization

Prepare fully supplemented T cell medium by adding cytokines and antibodies to the supplemented TexMACS Medium (see "Things to prepare in advance") as follows:

For T_H1 cell polarization (CytoBox T_H1, mouse):

• 10 ng/mL (60 U/mL) Mouse IL-12 
• 10 ng/mL (50 U/mL) Mouse IL-2 
• 10 µg/mL Anti-IL-4 pure – functional grade 

For T_H2 cell polarization (CytoBox T_H2, mouse):

• 10 ng/mL (200 U/mL) Mouse IL-4 
• 10 ng/mL (50 U/mL) Mouse IL-2 
• 10 µg/mL Anti-IFN-γ pure – functional grade

For T_H17 cell polarization (CytoBox T_H17, mouse):

• 20 ng/mL (10.000 U/mL) Mouse IL-6 
• 10 ng/mL (40 U/mL) Mouse IL-23 
• 10 ng/mL (8400 U/mL) Mouse IL-1β 
• 2 ng/mL (10 U/mL) Human TGF-β1
• 10 µg/mL Anti-IL-4 pure – functional grade 
• 10 µg/mL Anti-IFN-γ pure – functional grade 
• 10 µg/mL Anti-IL-2 pure – functional grad

▲ Note: Refer to "Things to prepare in advance" for conversion from U/mL to ng/mL. 

1. Resuspend loaded Anti-Biotin MACSiBead Particles thoroughly and transfer, for example, 40 μL (4×10⁶ loaded Anti-Biotin MACSiBead Particles) per 2×10⁶ cells (bead to cell ratio 2:1) to a suitable tube.
   ▲ Note: If unloaded MACSiBead Particles shall be used for negative control experiments, replace loaded Anti-Biotin MACSiBead Particles with unloaded Anti-Biotin MACSiBead Particles.
2. Add 1 mL of culture medium to the loaded Anti-Biotin MACSiBead Particles and centrifuge at 300×g for 5 minutes.
3. Aspirate supernatant and resuspend loaded Anti-Biotin MACSiBead Particles in, e.g., 1 mL of fully supplemented T cell medium including the cytokines and antibodies (from CytoBox) to a final concentration of 4×10⁶ Anti-Biotin MACSiBead™ Particles per mL.
4. Determine cell number of freshly isolated mouse naïve CD4⁺ T cells.
5. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cells at a density of 2×10⁶ cells per mL in fully supplemented T cell medium including the cytokines and antibodies (from CytoBox).
7. Transfer the desired amount of cells and the loaded Anti-Biotin MACSiBead Particles from step 4 and 7 at a ratio of 1:1 to a suitable cell culture vessel to a final density of 1×10⁶ cells per mL per cm² with 2 loaded Anti-Biotin MACSiBead Particles per cell (e.g., 2.5×10⁵ cells in 125 µL medium + 125 µl loaded Anti-Biotin MACSiBeads Particles per well of a 96-well plate). 
   ▲ Note: Depending on the cell number to be cultivated and differentiated per well, 6-, 12-, 24- or 48-well plates might be used. Please upscale reagents accordingly and refer to "Plate sizes for in vitro T cell polarization" to choose the appropriate cell culture dish.
8. Incubate at 37 °C and 5–10% CO₂ for up to 6 days. 
   ▲ Note: Inspect cultures daily and add fresh fully supplemented TexMACS Medium if required.
9. At day 2, gently pipette culture up and down to break up all cell clumps.
10. Split the cell culture every two days 1:4 or 1:2, depending on the proliferation of cells, and add fresh fully supplemented TexMACS Medium. 
11. After 6 days of cultivation, polarized T_H1, T_H2 or T_H17 cells can be further processed for downstream analysis. Refer to "Intranuclear staining of lineage-specific transcription factors" or "Intracellular staining of lineage-specific effector cytokines". T cells require a restimulation for further expansion or analysis of cytokine expression. See "Restimulation of polarized T cells and brefeldin A treatment" for details.

Note:

• For further flow cytometry analysis, it is recommended to remove the MACSiBead™ particles from the cell suspension.

1. Harvest cells and pool different wells of same condition.
2. Wash empty wells with cold PBE buffer to rinse out the remaining cells on the plate.
3. Determine cell number.
4. Wash cells with cold PBE buffer.
5. Resuspend cells in PBE buffer at a density of up to 2×10⁷ cells/mL and vortex thoroughly.
6. Place the tube in the magnetic field of the MACSiMAG™ Separator.
7. Allow the MACSiBead Particles to adhere to the wall of the tube for 4 minutes.
8. Keeping the tube in the magnet, carefully remove the supernatant containing the MACSiBead-depleted cells and place in a new tube.
9. Remove the tube from the separator and add buffer to the same volume as before.
10. Vortex sample, replace tube in the MACSiMAG Separator and repeat steps 7–8.
11. Collected cells can now be further processed as required.

Fixation and Permeabilization Solution (FoxP3 Staining Buffer Set)

To achieve the appropriate working concentration for safe fixation and permeabilization of cells, the Fixation/Permeabilization Solution 1 must be diluted 1:4 with the Fixation/Permeabilization Solution 2 (i.e., for 1×10⁶ cells use 0.25 mL of Fixation/Permeabilization Solution 1 plus  0.75 mL of Fixation/Permeabilization Solution 2).

Permeabilization Buffer (FoxP3 Staining Buffer Set)

▲ Note: Before dilution, make sure that the buffer does not contain any precipitates.

Transcription factor antibodies

• Analysis of T_H1 cells (e.g., T-bet; Anti-T-bet-PE, human and mouse; # 130-098-596)
• Analysis of T_H2 cells (e.g., Gata3 and T-bet; Anti-T-bet-PE, human and mouse; #130-098-596 and Anti-Gata3-APC, human and mouse; #130-100-649)
• Analysis of T_H17 cells (e.g., RORg(t); Anti-RORg(t)-APC, human and mouse; #130-103-838)

Notes:

• The recommended antibody dilution for all Anti-T-bet, Anti-Gata3 and Anti-RORg(T) conjugates is 1:11 for up to 1×10⁶ cells/100 μL of buffer for labeling of cells and analysis by flow cytometry.
• Use freshly prepared Fixation and Permeabilization Solution and Permeabilization Buffer from FoxP3 Staining Buffer Set  (#130-093-142) for cell fixation and permeabilization.

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend up to 1×10⁶ nucleated cells in 1 mL of cold, freshly prepared Fixation/Permeabilization Solution.
4. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
5. Wash cells by adding 1−2 mL of cold buffer per 1×10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
6. Wash cells by adding 1−2 mL of cold 1× Permeabilization Buffer per 1×10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
7. Resuspend up to 1×10⁶ nucleated cells in 100 μL of cold 1× Permeabilization Buffer.
   ▲ Note: For staining with several antibodies in this step, reduce the volume of 1× Permeabilization Buffer accordingly. For efficient permeabilization, the volume of 1× Permeabilization Buffer should be at least 30% of the overall staining volume.
8. Add 10 μL of each needed transcription factor antibody.
9. Mix well and incubate for 30 minutes in the dark in the refrigerator (2−8 °C).
10. Wash cells by adding 1−2 mL of cold 1× Permeabilization Buffer per 1×10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Aspirate supernatant completely.
11. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry or fluorescence microscopy.
    ▲ Note: Due to fixation and permeabilization, cells are smaller than viable cells. Thus, FSC/SSC settings of the flow cytometer may need to be adjusted.

1. Add 20 ng/mL PMA and 1µg/mL Ionomycin to cells and incubate for 3 hours at 37° C.
2. Add brefeldin A to a final concentration of 2 µg/mL to the cells and incubate for another 2 hours at 37° C.
3. Harvest cells, determine cell number and proceed to next step.

Notes:

• For analysis of T_H1 and T_H2 cells, use e.g., IFN-γ and IL-4; Anti-IFN-γ-APC,mouse; #130-102-340 and Anti-IL-4-PE, mouse; #130-102-435.
• For analysis of T_H17 cells, use e.g., IFN-γ and IL-17; Anti-IFN-γ-APC, mouse; #130-102-340 and Anti-IL-17A-PE, mouse; #130-102-344).
• The recommended antibody dilution of Anti-IFN-g-APC,mouse, Anti-IL-4-PE, mouse and Anti-IL-17A-PE, mouse for cell labeling and subsequent flow cytometry analysis 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).
• T cells require a restimulation prior to analysis of cytokine expression.
• After successful restimulation and brefeldin A treatment, intranuclear transcription factor and intracellular cytokine staining can be combined for certain cytokine/transcription factor combinations. In this case, please follow the protocol steps "Restimulation of polarized cells and brefeldin A treatment", "Intracellular staining of lineage-specific effector cytokines", and "Intranuclear staining of lineage-specific transcription factors" in this order.
  
• Anti-IL-4 staining cannot be combined with intranuclear staining using the FoxP3 Staining Buffer Set.
• Use Inside Fix and Inside Perm from Inside Stain Kit (#130-090-477) for cell permeabilization and fixation.

1. Wash up to 1×10⁶ cells by adding 1–2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
2. (Optional) Stain cell surface antigens that are sensitive to fixation with appropriate antibodies according to the manufacturer's recommendations. Then wash cells by adding 1–2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend up to 1×10⁶ cells in 250 μL of buffer.
4. Add 250 μL of Inside Fix (from Inside Stain Kit). Mix well and incubate for 20 minutes in the dark at room temperature.
5. Centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
6.  Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
   ▲ Note: Fixed cells may be stored in azide containing buffer at 2–8 °C for up to 1 week.
7. (Optional) Stain cell surface antigens that are sensitive to permeabilization with appropriate antibodies. Then wash cells by adding 1–2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
8. Wash cells by adding 1 mL of Inside Perm (Inside Stain Kit) and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
9. Resuspend cells in 45 μL of Inside Perm. Add 5 μL of the antibody.
   ▲ Note: For staining with several antibodies in this step, reduce the volume of Inside Perm accordingly. For efficient permeabilization, the volume of Inside Perm should be at least 30% of the overall staining volume.
10. Mix well and incubate for 10 minutes in the dark at room temperature.
11. Wash cells by adding 1 mL of Inside Perm and centrifuge at 300×g for 5 minutes. Aspirate supernatant carefully.
12. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry or fluorescence microscopy. Store cells at 2–8 °C in the dark until analysis. Mix well before flow cytometric acquisition.
    ▲ Note: Samples may be stored at 2–8 °C in the dark for up to 24 hours.
    ▲ Note: Do not use propidium iodide (PI) or 7AAD staining.

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