PBMCs, isolated from HCWs (n=7) and RA patients (n=15), were thawed, counted, assessed for viability, and rested for 2-4 hours at 37°C in RPMI supplemented with 1% L-glutamine, 1% penicillin/streptomycin (Euroclone S.p.A, Italy), and 10% heat-inactivated FBS. For antigen-specific T-cell stimulation, PBMCs were seeded at a concentration of 2.5 × 106 cells/mL in a final volume of 200 µL in a 96-multiwell flat-bottom plate (COSTAR, Sigma Aldrich), and stimulated with spike peptide pool at 1 µg/mL or Staphylococcal Enterotoxin B (SEB) at 200 ng/mL, used as a positive control. We added anti-CD28 and anti-CD49d monoclonal antibodies (BD Biosciences San Jose, USA) to co-stimulate cells at a final concentration of 1 µg/mL each. After 1h of incubation at 37°C (5% CO2), a Golgi plug (BD Biosciences) at 1 µL/mL was added to cell cultures to inhibit cytokine secretion and to allow intracellular molecule detection by flow cytometry. After 16-24 h, cells were stained as described in the following.
Anti cd28 and anti cd49d monoclonal antibodies
Manufactured by BD
Sourced in United States
Anti-CD28 and anti-CD49d monoclonal antibodies are laboratory reagents used in immunological research. They target the surface molecules CD28 and CD49d, which are involved in T cell activation and trafficking. These antibodies can be used to stimulate or inhibit various immune cell functions in in vitro experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Euroclone (2 mentions)
L glutamine, by Euroclone (2 mentions)
Golgiplug, by BD (2 mentions)
Brefeldin a bfa, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Ctl immunospot reader, by Cellular Technology (1 mentions)
Sepmate 15, by STEMCELL (1 mentions)
Sepmate 50, by STEMCELL (1 mentions)
Fitc anti il 2 and pe anti ifnγ, by BD (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cpt tube, by BD (1 mentions)
Benzonase, by Merck Group (1 mentions)
Rpmi 1640 medium, by Biowest (1 mentions)
Monensin, by BioLegend (1 mentions)
7 protocols using anti cd28 and anti cd49d monoclonal antibodies
1
PBMC Stimulation and Flow Cytometry
2
Intracellular Cytokine Staining of PBMC
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We isolated fresh PBMC using Ficoll density gradient centrifugation, and 1x106 cells/ml were cultured overnight with the single stimuli and the following costimuli as anti-CD28 and anti-CD49d monoclonal antibodies at 2 μg/ml each (BD Bioscence) at 37°C and 5% CO2 in 10% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria) in RPMI-1640 (Gibco, CA, USA). To prevent cytokine secretion the BD GolgiPlug was added after 1 hr of stimulation. After 16 hrs of incubation the ICS was performed. Unstimulated PBMC provided as a negative control. PBMC were first stained with mAb for surface markers, fixed in 4% paraformaldehyde and permeabilized with PBS-1% BSA -0.5% saponin -0.1% NaN3 and then stained with mAb for intracellular cytokines. Cells were fixed again in 2% paraformaldehyde, and at least 200,000 lymphocytes were acquired using a FACSCanto II flow cytometer (BD Biosciences).
3
Human IFN-γ/IL-4 Dual Color ELISPOT Assay
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Human IFN-γ/IL-4 dual color ELISPOT assays were performed according to the manufacturer’s instructions (Abcam, Cambridge, UK). Briefly, polyvinylidene difluoride-backed microtiter plates (MSIP, Millipore, Temecula, CA, USA) were treated with 25 μl of 35% methanol for 30 s and washed 3 times with sterile PBS. Plates were coated with IFN-γ/IL-4 capture antibodies at 4 °C overnight. Human PBMCs (5 × 105 cells per well) were stimulated with 10 μg/mL ScaA, TSA56 or 9-mer epitope peptides (1 μM) in the presence of co-stimulatory anti-CD28 and anti-CD49d monoclonal antibodies (BD Pharmingen) at 37 °C for 18 h. The wells were incubated further with a secondary anti-IFN-γ FITC-conjugated antibody and biotinylated anti-IL-4 antibody at room temperature for 90 min, followed by incubation with anti-FITC HRP and streptavidin-alkaline phosphatase (AP) conjugates for 1 h. Immunospots were developed by sequential incubation of wells with an AEC or BCIP/NBT substrate and were counted using a CTL ImmunoSpot reader (Cellular Technology, Cleveland, OH, USA). The count settings (intensity, gradient and size of spot) were manually confirmed by the naked eye in each experimental set.
4
PBMC Isolation and Stimulation for COVID-19 Vaccine Response
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Peripheral blood mononuclear cells (PBMCs) from a small subset of the vaccinated individuals (8 patients with MS and 7 HCWs) were isolated on density gradient centrifugation (SepMate-50 cat#85460 or SepMate-15 cat#85420, StemCell Technologies) according to manufacturer's procedure. The 7 HCWs, used as control group, were employed as controls in another publication.27 (link) All samples were frozen in heat-inactivated fetal bovine serum (FBS; Euroclone SpA) with 10% DMSO and stored in liquid nitrogen. PBMCs were thawed, counted, assessed for viability, and rested for 2–4 hours at 37°C in RPMI+10% FBS prior to further use. Complete medium was freshly prepared as follows: RPMI-1640, 10% FBS, 1% l -glutamine, and 1% penicillin/streptomycin (Euroclone SpA). Cells were seeded at a concentration of 2.5 × 106 cells/mL in a 96-multiwell flat-bottom plate (COSTAR; Sigma Aldrich) and stimulated with spike peptide pool at 1 µg/mL or staphylococcal enterotoxin B (SEB) at 200 ng/mL, as a positive control. Anti-CD28 and anti-CD49d monoclonal antibodies (BD Biosciences) were added at 2 µg/mL to costimulate cells. After 1 hour of incubation at 37°C (5% CO2), 1 µL/mL of Golgi Plug (BD Biosciences) was added to cell cultures to inhibit cytokine secretion. Following an incubation of 16–24 hours, cells were stained as described below.
5
Phenotyping Antigen-Specific T Cells
6
PBMC Isolation, Cryopreservation, and Antigen Stimulation
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PBMCs were isolated using CPT tubes (BD Biosciences). Afterwards, cells were cryopreserved and stored in liquid nitrogen for later flow cytometry analyses. Cryopreserved PBMCs were thawed and rested during 2 h in a humidified incubator at 37°C with 5% CO2 in RPMI 1,640 medium (Biowest, Nuaillé, France) containing 10% of heat-inactivated fetal calf serum (FCS) with benzonase (Sigma, St. Louis, MO, USA; final concentration 10 U/mL). PBMCs from each patient were stimulated overnight at 37°C with 5% CO2 with the recombinant proteins ESAT-6/CFP-10 (Lionex Diagnostics and Therapeutics, Braunschweig, Germany; final concentration 2 μg/mL for each antigen) and PPD (Statens Serum Institut, Copenhagen, Denmark; final concentration 10 μg/mL). The staphylococcal enterotoxin B (SEB; Sigma; final concentration 2.5 μg/mL) was used as a positive control. A negative control without stimulation was also included. Cells were also co-stimulated with anti-CD28 and anti-CD49d monoclonal antibodies (BD Bioscience; final concentration 2 μg/mL each). After 2 h of incubation, Brefeldin A (BFA; Sigma; final concentration 3 μg/mL) was added into the culture media to inhibit the intracellular vesicular transport. Then, PBMCs were left in the incubator overnight.
7
PBMC Stimulation with Mycobacterial Antigens
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Samples included in the study were stimulated for 16 h separately with two mycobacterial antigen mixes: (i) recombinant proteins ESAT-6/CFP-10 (Lionex Diagnostics and Therapeutics, Braunschweig, Germany; final concentration 2 μg/ml each) and (ii) PPD (AJVaccines, Copenhagen, Denmark; final concentration 10 μg/ml). A positive control consisting of staphylococcal enterotoxin B (SEB, Sigma, final concentration of 2 μg/ml) and a negative control without stimulation were also included for each sample. One million (106) PBMCs were used in each stimulation condition, adding as co-stimulators anti-CD28 and anti-CD49d monoclonal antibodies (BD Bioscience; final concentration 1 μg/ml each). After a 2 h incubation at 37°C in a 5% CO2 atmosphere, Brefeldin A (BFA; Sigma; final concentration 3 μg/ml) and Monensin (BioLegend, San Diego, USA, final concentration 1X) were added to inhibit intracellular vesicular transport. Cells were then incubated overnight before starting the staining procedure.
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