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Its 1 solution

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The ITS+1 solution is a laboratory reagent that provides a defined, serum-free supplement for cell culture media. It contains insulin, transferrin, and selenium, which are essential components for supporting cell growth and proliferation.

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Lab products found in correlation

Gentamycin, by Merck Group (9 mentions) Sodium pyruvate, by Lonza (9 mentions) Bovine serum albumin (bsa), by Merck Group (9 mentions) Rbc lysis buffer, by Merck Group (7 mentions) Non essential amino acids (neaa), by Lonza (6 mentions) Hepes, by Lonza (5 mentions) Nonessential amino acids 0.2, by Lonza (3 mentions) Red blood cell lysis buffer, by Merck Group (2 mentions) Hepes, by GE Healthcare (1 mentions)

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ITS+1 (18 citations) ITS solution (15 citations)

9 protocols using its 1 solution

1

Reactivation and Expansion of Antigen-Specific T Cells

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The mouse spleen was collected in 5% fetal bovine serum (FBS) in RPMI-1640 (RPMI) supplemented with 10 mg/ml gentamycin (Sigma), 10 mM HEPES, 1 mM sodium pyruvate (Lonza BioWhittaker, Walkersville, MD), non-essential amino acids 0.2% (Lonza BioWhittaker, Walkersville, MD). The spleen was then made into a single-cell suspension that was depleted of red blood cells using red blood cell lysis buffer (Sigma). Serum-free media (SFM) that consisted of RPMI with 1% ITS+1 solution (Sigma) and 0.1% bovine serum albumin (BSA) (Sigma) was used to resuspend the red blood cell-depleted splenocytes and IRBP was added at 50 mg/ml for 48 hours at 37 °C and 5% CO2 to reactivate antigen-specific T cells. Following the reactivation cells were collected for adoptive transfer into recipient mice or stained for flow cytometry analysis.
Human PBMCs were activated through CD3 and CD28 stimulation. A 24-well plate was coated with anti-CD3 (10 mg/ml) and anti-CD28 (10 mg/ml) in sterile phosphate-buffered saline (PBS) (250 μl/per well) by overnight incubation at 4 °C. Wells were loaded with 2 - 10 × 106 PBMCs ± CGS21680 (1 mM) in SFM and incubated for 48 hours at 37 °C and 5% CO2. After the 48-hour incubation, cells were collected and stained for flow cytometry analysis. Cells cultured with anti-CD3 and anti-CD28 or in media alone were used as controls.
Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.
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2

Isolation of Suppressor Antigen-Presenting Cells

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The spleens from mice that recovered from EAU (day 85–90 after immunization) or unimmunized mice were collected into 5% FBS in RPMI supplemented with 10 μg/mL Gentamycin (Sigma, St. Louis, MI), 10 mM HEPES, 1 mM sodium pyruvate (BioWhittaker, Basel, Switzerland), nonessential amino acids 0.2% (BioWhit-taker). Spleen cells were made into a single cell suspension that was subsequently depleted of RBCs with RBC lysis buffer (Sigma, St Louis, MO). The RBC-free spleen cells were then separated to obtain adherent APC that were then cultured in serum-free media (SFM) with 1 ng/mL α-MSH for 48 h at 37°C and 5% CO2. SFM consisted of RPMI-1640 with 1% ITS+1 solution (Sigma) and 0.1% BSA (Sigma). The cultured APC were then stained and sorted to obtain a pure population of suppressor APC.
Muhammad F., Trivett A., Wang D, & Lee D.J. (2019). Tissue-specific production of MicroRNA-155 inhibits melanocortin 5 receptor-dependent suppressor macrophages to promote experimental autoimmune uveitis. European journal of immunology, 49(11), 2074-2082.
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3

Reactivation of Antigen-Specific T Cells

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A single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO) was made from the spleens collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES (GE Healthcare), 1 mM Sodium Pyruvate (BioWhittaker), Nonessential Amino Acids 0.2% (BioWhittaker). Serum free media (SFM) consisting of RPMI-1640 with 1% ITS+1 solution (Sigma) and 0.1% BSA (Sigma) was used to resuspend the spleen cells with IRBP (50 μg/mL) or α-CD3 (clone 2C11, Biolegend). The spleen cells were then incubated for 48 h at 37°C and 5% CO2 to reactivate antigen specific T cells. After the reactivation cells were collected for flow cytometry analysis or adoptive transfer (1 × 106) into recipient mice.
Muhammad F., Avalos P.N., Mursalin M.H., Ma J.X., Callegan M.C, & Lee D.J. (2020). Kallistatin Attenuates Experimental Autoimmune Uveitis by Inhibiting Activation of T Cells. Frontiers in Immunology, 11, 975.
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4

Reactivation of Antigen-Specific T Cells

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Spleens were collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES, 1 mM Sodium Pyruvate (BioWhittaker), Nonessential Amino Acids 0.2% (BioWhittaker) and made into a single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO). The spleen cells were resuspended in serum free media (SFM) and IRBP was added at 50 μg/mL for 48 hours at 37°C and 5% CO2 to reactivate antigen specific T cells. SFM consisted of RPMI-1640 with 1% ITS+1 solution (Sigma) and 0.1% BSA (Sigma). Following the reactivation supernatants were collected and analyzed and/or cells were collected for adoptive transfer into recipient mice.
Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.
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5

Adoptive Transfer of Antigen-Specific T Cells for Uveitis

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Post-EAU spleens (day 90–100 after immunization for EAU) were collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES, 1 mM Sodium Pyruvate (BioWhittaker), Nonessential Amino Acids 0.2% (BioWhittaker) and made into a single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO). The spleen cells were resuspended in serum free media (SFM) and IRBP was added at 50 μg/mL for 48 hours at 37 °C and 5% CO2 to reactivate antigen specific T cells. SFM consisted of RPMI-1640 with 1% ITS + 1 solution (Sigma) and 0.1% BSA (Sigma). PD-1 neutralizing antibody (clone J43, ebioscience) was added during reactivation to block the PD-1/PD-L1 pathway where indicated. Following reactivation, cells were collected for adoptive transfer of 1 × 106 cells by tail vein injection into recipient mice at the same time as immunization for EAU.
Muhammad F., Wang D., Montieth A., Lee S., Preble J., Foster C.S., Larson T.A., Ding K., Dvorak J.D, & Lee D.J. (2019). PD-1+ melanocortin receptor dependent-Treg cells prevent autoimmune disease. Scientific Reports, 9, 16941.
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6

Reactivation and Expansion of Antigen-Specific T Cells

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The mouse spleen was collected in 5% fetal bovine serum (FBS) in RPMI-1640 (RPMI) supplemented with 10 mg/ml gentamycin (Sigma), 10 mM HEPES, 1 mM sodium pyruvate (Lonza BioWhittaker, Walkersville, MD), non-essential amino acids 0.2% (Lonza BioWhittaker, Walkersville, MD). The spleen was then made into a single-cell suspension that was depleted of red blood cells using red blood cell lysis buffer (Sigma). Serum-free media (SFM) that consisted of RPMI with 1% ITS+1 solution (Sigma) and 0.1% bovine serum albumin (BSA) (Sigma) was used to resuspend the red blood cell-depleted splenocytes and IRBP was added at 50 mg/ml for 48 hours at 37 °C and 5% CO2 to reactivate antigen-specific T cells. Following the reactivation cells were collected for adoptive transfer into recipient mice or stained for flow cytometry analysis.
Human PBMCs were activated through CD3 and CD28 stimulation. A 24-well plate was coated with anti-CD3 (10 mg/ml) and anti-CD28 (10 mg/ml) in sterile phosphate-buffered saline (PBS) (250 μl/per well) by overnight incubation at 4 °C. Wells were loaded with 2 - 10 × 106 PBMCs ± CGS21680 (1 mM) in SFM and incubated for 48 hours at 37 °C and 5% CO2. After the 48-hour incubation, cells were collected and stained for flow cytometry analysis. Cells cultured with anti-CD3 and anti-CD28 or in media alone were used as controls.
Peters K., McDonald T., Muhammad F., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2023). A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice. Mucosal immunology, 16(4), 422-431.
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7

Reactivation of Antigen-Specific T Cells

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Spleens were collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES, 1 mM Sodium Pyruvate (BioWhittaker), Nonessential Amino Acids 0.2% (BioWhittaker) and made into a single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO). The spleen cells were resuspended in serum free media (SFM) and IRBP was added at 50 μg/mL for 48 hours at 37°C and 5% CO2 to reactivate antigen specific T cells. SFM consisted of RPMI-1640 with 1% ITS+1 solution (Sigma) and 0.1% BSA (Sigma). Following the reactivation, supernatants were collected and analyzed and/or cells were collected for adoptive transfer into recipient mice.
In some experiments antigen presenting cells (APC) and T cells were cultured from different strains. APC were collected by incubating splenocytes in SFM at 37°C and 5% CO2 for 90 minutes in tissue culture plates, washed twice, and adherent cells were scraped off of the plastic in ice cold SFM, and plated at 4 x 105 cells per well. CD3 enriched T cells were obtained from post-EAU spleens using a CD3 enrichment column (R&D Systems), added to the sorted APC at 8 x 105 cells per well with 50 μg IRBP peptide, and cultured at 37°C 5% CO2 for 48 hours. After 48 hours, T cells and APC were collected, and washed in PBS. Following the reactivation, cells were collected for adoptive transfer into recipient mice.
McDonald T., Muhammad F., Peters K, & Lee D.J. (2021). Combined Deficiency of the Melanocortin 5 Receptor and Adenosine 2A Receptor Unexpectedly Provides Resistance to Autoimmune Disease in a CD8+ T Cell-Dependent Manner. Frontiers in Immunology, 12, 742154.
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8

Reactivation of Antigen-Specific T Cells

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Spleens were collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES, 1 mM sodium pyruvate (BioWhittaker), nonessential amino acids 0.2% (BioWhittaker), and made into a single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO). The spleen cells were resuspended in serum free media (SFM) and IRBP residues 1-20 (IRBP) was added at 50 μg/ml for 48 h at 37°C and 5% CO2 to reactivate Ag-specific T cells. SFM consisted of RPMI-1640 with 1% ITS+1 solution (Sigma) and 0.1% BSA (Sigma). Following the reactivation, supernatants were collected and analyzed and/or cells were collected for adoptive transfer into recipient mice.
Muhammad F.Y., Peters K., Wang D, & Lee D.J. (2019). Exacerbation of autoimmune uveitis by obesity occurs through the melanocortin 5 receptor. Journal of leukocyte biology, 106(4), 879-887.
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9

Reactivation of Antigen-Specific T Cells

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Spleens were collected into 5% FBS in RPMI supplemented with 10 μg/ml Gentamycin (Sigma), 10 mM HEPES, 1 mM Sodium Pyruvate (BioWhittaker), Nonessential Amino Acids 0.2% (BioWhittaker) and made into a single cell suspension that was depleted of red blood cells using RBC lysis buffer (Sigma, St Louis, MO). The spleen cells were resuspended in serum free media (SFM) and IRBP peptide was added at 50 μg/mL for 48 hours at 37 °C and 5% CO2 to reactivate antigen specific T cells. SFM consisted of RPMI-1640 with 1% ITS + 1 solution (Sigma) and 0.1% BSA (Sigma). Following the in vitro reactivation supernatants were collected and analyzed and/or cells were collected for adoptive transfer into recipient mice. Recipient mice were immunized for EAU on the same day as receiving 1 × 106 reactivated spleen cells.
Lee D.J., Preble J., Lee S., Foster C.S, & Taylor A.W. (2016). MC5r and A2Ar Deficiencies During Experimental Autoimmune Uveitis Identifies Distinct T cell Polarization Programs and a Biphasic Regulatory Response. Scientific Reports, 6, 37790.
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