After 24 h, unlabeled and ferumoxytol-labeled hUCB-MSCs were washed with DPBS and detached using 0.25 % trypsin (Sigma-Aldrich). The surface antigens of unlabeled and ferumoxytol-labeled hUCB-MSCs were phenotyped by staining the cells with FITC, PE, or APC-coupled antibodies for 15 min at RT. Anti-human antibodies against the following proteins were used for fluorescence-activated cell sorting (FACS): CD14, CD45, CD73, CD90, CD105, and HLA-DR (BD Pharmingen, San Jose, CA, USA). IgG1 and IgG2a (BD Pharmingen) were used as the corresponding mouse isotype controls. Labeled cells were washed with DPBS, fixed with 1 % paraformaldehyde (PFA; Biosesang, Gyeonggi-do, Republic of Korea), and analyzed by the MACSQuant® Analyzer (Miltenyi Biotec, San Diego, CA, USA).
Igg1 and igg2a
Manufactured by BD
IgG1 and IgG2a are immunoglobulin subclass proteins used in laboratory analysis and research. IgG1 and IgG2a are the primary antibody types found in mammalian blood and play a crucial role in the immune response. These proteins can be used to detect and quantify specific antibodies or antigens in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hla dr, by BD (2 mentions)
Cd105, by BD (2 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Trypsin, by Merck Group (1 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
Tetramethylbenzidine tmb, by Thermo Fisher Scientific (1 mentions)
P nitrophenylphosphate p npp solution, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cd146, by BD (1 mentions)
Cd271, by BD (1 mentions)
Cd79a, by BD (1 mentions)
4 protocols using igg1 and igg2a
1
Characterization of Ferumoxytol-Labeled hUCB-MSCs
2
ELISA for Autoantibody Detection
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Example 17
ELISA plates (Nunc Immunoplate MaxiSorp; Fisher Scientific) were coated with 3 μg/ml MP4, 10 μg/ml MOG:35-55 or 3 μg/ml ovalbumin (OVA) in bicarbonate buffer overnight at 4° C. The plates were blocked for 2 hours with phosphate-buffered saline (PBS), containing 0.05 Tween 20 (PBST) and 1% BSA at room temperature. Subsequently, serum samples diluted in PBST/BSA, were added to the plate for overnight incubation at 4° C. Secondary antibodies used were biotinylated rat anti-mouse IgG (eBioscience, San Diego, Calif.), IgG1 and IgG2a (BD-Pharmingen, San Jose, Calif.). Streptavidin-AP or Streptavidin-HRP served as tertiary reagents (DakoCytomation, Glostrup, Denmark), both diluted at 1:1000 in PBST/BSA. Plates were developed with 100 μl/well of freshly prepared p-nitrophenylphosphate (p-NPP) solution (Sigma-Aldrich) or tetramethylbenzidine (TMB) (eBioscience).
3
Phenotypic Characterization of hMSCs
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hMSCs at P3 or P4 were expanded in T175 until they reached confluence. The cells were trypsinized and incubated for 30 min in blocking buffer consisting of 17% bovine serum albumin (Sigma – Cat. No.: F7524) in PBS followed by incubation with FITC- or PE-conjugated mouse anti-human antibodies for 30 min at 4 °C in the dark. The samples were then washed three times with a washing buffer consisting of 3% bovine serum albumin in PBS. The expression levels were analyzed using FACSAria flow cytometer (BD Bioscience). For phenotypic characterization the following antibodies were used: CD90, CD73, CD146, CD105, CD271, CD34, CD14, CD79a, HLA-DR, CD45 and IgG1 and Ig G2a as isotype controls (all from BD Pharming).
4
Quantifying Antibody Levels in Immunized Mice
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Prior to the respiratory challenge, sera from immunized mice were used to quantify the levels of antibodies by enzyme-linked immunosorbent assay (ELISA)40 (link),63 (link). Multiwell plates were coated with C. muridarum EB (1 μg/well) or rMOMP (0.1 μg/well) and incubated with serial dilutions of preimmune and immune sera, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (KPL, catalog number 474–1806 diluted 1:4000), and IgG1 and IgG2a (BD Pharmingen, catalog numbers 559626 and 553391, respectively, diluted 1:1000) antibodies. Chromogenic substrate detection at OD405 was performed using an EIA reader (Labsystem Multiscan, Helsinki, Finland) and geometric mean titers (GMTs) were expressed as the reciprocal of the dilution40 (link),63 (link).
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