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Fix and perm media a and b

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Fix and Perm media A and B are laboratory reagents used in sample preparation and processing. Media A is a fixative solution, while Media B is a permeabilization solution. These products are designed to prepare biological samples for further analysis, such as flow cytometry or microscopy. The core function of these media is to preserve cellular structures and allow access to intracellular components.

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Fix and Perm A and B Perm A and B Fix and Perm Fix/Perm Perm B

4 protocols using fix and perm media a and b

1

Antigen-Specific CD8+ T Cell Assay

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Spleen and skin cell populations were cultured in 96-well round-bottom plates at 106 cells per well in complete CR10 media (RPMI, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.1 mM non-essential amino acids). 106 irradiated splenocytes isolated from naive, congenic C57BL/6 mice were added to the wells with or without 10 μM of the HSV-1 gB498-505 peptide and 1 μg/mL anti-CD28 antibody. 10 μg/mL brefeldin A was added to the wells at the start of culture. After a 4 hour incubation at 37°C, cells were placed on ice, washed and stained with antibodies directed against CD3, CD8, CD45.1, CD45.2, CD44 and fixable viability dye. Cells were fixed and permeabilized using Fix and Perm media A and B (Thermo Fisher) and stained intracellularly with anti-IFN-γ (XMG1.2).
Bromley S.K., Akbaba H., Mani V., Mora-Buch R., Chasse A.Y., Sama A, & Luster A.D. (2020). CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response. Cell reports, 32(9), 108085.
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2

Antigen-Specific CD8+ T Cell Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Spleen and skin cell populations were cultured in 96-well round-bottom plates at 106 cells per well in complete CR10 media (RPMI, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.1 mM non-essential amino acids). 106 irradiated splenocytes isolated from naive, congenic C57BL/6 mice were added to the wells with or without 10 μM of the HSV-1 gB498-505 peptide and 1 μg/mL anti-CD28 antibody. 10 μg/mL brefeldin A was added to the wells at the start of culture. After a 4 hour incubation at 37°C, cells were placed on ice, washed and stained with antibodies directed against CD3, CD8, CD45.1, CD45.2, CD44 and fixable viability dye. Cells were fixed and permeabilized using Fix and Perm media A and B (Thermo Fisher) and stained intracellularly with anti-IFN-γ (XMG1.2).
Bromley S.K., Akbaba H., Mani V., Mora-Buch R., Chasse A.Y., Sama A, & Luster A.D. (2020). CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response. Cell reports, 32(9), 108085.
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3

Intracellular Cytokine Staining of Immune Cells

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For direct intracellular cytokine staining, spleen and a 1 cm2 piece of skin at the peptide challenge site were harvested and placed on ice. Skin was minced and digested in skin digestion buffer containing 10 μg/mL Brefeldin A as described above. Recovered leukocytes were then stained in buffer containing Brefeldin A with antibodies directed against CD3, CD8, CD45.1, CD45.2, CD44 and fixable viability dye. Cells were fixed and permeabilized using Fix and Perm media A and B (Thermo Fisher) and stained intracellularly with anti-IFN-γ (XMG1.2).
Bromley S.K., Akbaba H., Mani V., Mora-Buch R., Chasse A.Y., Sama A, & Luster A.D. (2020). CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response. Cell reports, 32(9), 108085.
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4

Intracellular Cytokine Staining of Immune Cells

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For direct intracellular cytokine staining, spleen and a 1 cm2 piece of skin at the peptide challenge site were harvested and placed on ice. Skin was minced and digested in skin digestion buffer containing 10 μg/mL Brefeldin A as described above. Recovered leukocytes were then stained in buffer containing Brefeldin A with antibodies directed against CD3, CD8, CD45.1, CD45.2, CD44 and fixable viability dye. Cells were fixed and permeabilized using Fix and Perm media A and B (Thermo Fisher) and stained intracellularly with anti-IFN-γ (XMG1.2).
Bromley S.K., Akbaba H., Mani V., Mora-Buch R., Chasse A.Y., Sama A, & Luster A.D. (2020). CD49a Regulates Cutaneous Resident Memory CD8+ T Cell Persistence and Response. Cell reports, 32(9), 108085.
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