Cryopreserved cells were thawed, counted, and resus-pended in complete medium (R10) RPMI 1640 (Gibco; Grand Island, NY) supplemented with 10% fetal bovine serum (Gemini Bio-Products; West Sacramento, CA) and 5% Pen-Strep-Glut (Gibco; Grand Island, NY). Cell suspensions at 2 × 106 cells/mL were incubated overnight at 37 °C, 5% CO2 in 50 mL tubes. The IFN-γ ELISpot assay was performed using Mabtech pre-coated plates (Mabtech; Mariemont, Ohio); Mabtech biotinylated anti-IFN-γ 7-B6-1 monoclonal antibody (Ab) was used as the detecting Ab; NovaRED substrate (Vector Laboratories; Burlingame, CA) was used to reveal the presence of spots. Spots formed by IFN-γ-secreting cells were counted with an automated ImmunoSpot plate reader (Cellular Technologies, Cleveland Ohio), and results are presented as spot-forming cells (SFC) as indicated in each figure. For all samples, the cells were evaluated for their functionality using the CEF and CMVpp65 peptide pools at the concentration of 1 µg/mL. For the HIV-1 seropositive samples, additional testing was performed using the PTE peptides. This assay was performed following Good Clinical Laboratory Procedures guidelines; detailed Standard Operating Procedures are available upon request.
R10 rpmi 1640
Manufactured by Thermo Fisher Scientific
RPMI 1640 is a cell culture medium used to support the growth of a variety of cell types, including human and animal cells. It provides the necessary nutrients and growth factors for cell maintenance and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Immunospot plate reader, by Cellular Technology (1 mentions)
Pen strep glut, by Thermo Fisher Scientific (1 mentions)
Novared substrate, by Vector Laboratories (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Peflacin, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids solution, by Thermo Fisher Scientific (1 mentions)
2 mercaptoetanol, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Mouse cd11c microbeads ultrapure, by Miltenyi Biotec (1 mentions)
Mouse b cell isolation kit, by Miltenyi Biotec (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
70 mm cell strainer, by Corning (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by AppliChem (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
3 protocols using r10 rpmi 1640
1
IFN-γ ELISpot Assay for Immune Functionality
2
Isolation of Murine Immune Cells
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Two weeks after the last immunization, mice were euthanized under general anesthesia with a mixture of ketamine 300mg/kg and xylazine 30mg/kg via i.p. and spleens/ draining lymph nodes were removed aseptically. Single cell suspensions were obtained after red blood cell lysis with ammonium chloride potassium (ACK) solution. Cells were then resuspended in R-10 (RPMI 1640 supplemented with 10% of fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 10 mM Hepes (Gibco), 1mM sodium pyruvate (Gibco), 1% v/v non-essential aminoacids solution (Gibco), 40 μg/mL of Gentamicin, 20 μg/mL of Peflacin and 5x10-5M 2- mercaptoetanol (Gibco). The viability of cells was evaluated using 0.2% Trypan Blue exclusion dye to discriminate between live and dead cells. Cell concentration was estimated with the aid of a cell counter (Countess- Invitrogen) and adjusted in cell culture medium.
3
Isolation of Murine Splenic Immune Cells
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To isolate splenic immune cells, murine spleens were cut into small pieces and digested with 0.1 mg/mL collagenase D (Sigma-Aldrich, 11088866001) and 0.05 mg/mL DNase (Sigma-Aldrich, D5025-150KU) for 10 minutes at 37 C. EDTA (Applichem, A4892.1000) was added at a concentration of 0.01 mol/L, followed by a second incubation step at 37 C for 5 minutes. The splenocytes solution was smashed through a 70-mm cell strainer (Corning, 431751). Red blood cells were lysed with cell lysis buffer (BD, 555899). Mouse CD11c UltraPure microbeads (Miltenyi Biotec, 130-108-338), mouse CD8a þ T Cell Isolation Kit (Miltenyi Biotec, 130-104-075) or the Mouse B Cell Isolation Kit (Miltenyi Biotec, 130-090-862) were used according to the manufacturer's instructions to isolate different immune cell populations from the splenocytes suspension. For subsequent in vitro assays with splenic immune cells, cells were resuspended in R10 (RPMI1640; Gibco, 31870-025) supplied with 10% FBS (Gibco, 16140), 1% penicillin-streptomycin (P/S; Gibco, 11548876), 1% L- glutamine (Gibco, 25030-024), 1% sodium-pyruvate (Gibco, 11360-039), 1% nonessential amino acids (Gibco, 11140-035), and 50 mmol/L b-Mercaptoethanol (Gibco, 31350-010).
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