Binding of human antibodies to pig cells was measured by flow cytometry using the relative geometric mean (rGM), as previously described (11 (link)). Briefly, pRBCs were separated from whole blood, washed x3 with phosphate-buffered saline (PBS), and centrifuged at 700g for 5min at 4°C. The washed RBCs were suspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 1% bovine serum albumin). pPBMCs were isolated using Ficoll (HaoYang, Tianjin, China) and suspended in FACS buffer for IgM/IgG binding assays. The isolated pRBCs (5x105/tube) and pPBMCs (5x105/tube) were incubated with heat-inactivated human serum at 4°C for 30min, respectively, and the final serum concentration was 20%. After incubation, cells were washed with PBS to remove unbound antibodies and were blocked with 10% goat serum for 15min at 4°C. After further washing with PBS, anti-human IgM or anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) (IgG: concentration 1:1000 for pRBCs and pPBMCs; IgM: concentration 1:1600 for pRBCs and pPBMCs) was added, and the cells were incubated for 30min at 4°C. After washing with PBS, 100μL PBS buffer was added. Flow cytometry was carried out using BD FACSCelesta (Becton Dickinson, San Jose, CA, USA).
Anti human igm
Manufactured by Jackson ImmunoResearch
Sourced in United States, Cameroon
Anti-human IgM is a laboratory reagent used to detect and quantify human immunoglobulin M (IgM) in biological samples. It is a highly specific antibody that binds to IgM, allowing for the identification and measurement of IgM levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti human igg, by Jackson ImmunoResearch (2 mentions)
Facscelesta, by BD (1 mentions)
24 well insert, by Corning (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Anti cd69 pe, by BD (1 mentions)
Cpg odn, by Thermo Fisher Scientific (1 mentions)
Anti ikkβ, by Cell Signaling Technology (1 mentions)
Z vrpr fmk, by Enzo Life Sciences (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Anti malt1, by Santa Cruz Biotechnology (1 mentions)
Anti phospho ikkβ, by Cell Signaling Technology (1 mentions)
Anti a20, by Santa Cruz Biotechnology (1 mentions)
Anti irak1, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Etoposide, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Complete mini, by Roche (1 mentions)
Sector s 600, by MSD (1 mentions)
Cardiolipin cl, by Merck Group (1 mentions)
9 protocols using anti human igm
1
Antibody Binding to Pig Cells
2
Activation and Migration of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell migration assay was conducted using 24-well insert (Costar) with 5-µm pores. PBMCs from normal, healthy donors were activated with either CPG-ODN, a synthetic TLR9 agonist (Invitrogen), or Phytohemagglutinin (PHA) (Sigma) or anti-human IgM (Jackson Immuno Research) and anti CD40 monoclonal antibody, clone B-B20 (BioSite).
The activation status of the cells was then investigated by measuring CD69 expression on CD19+ and CD3+ PBMCs by Flow cytometry prior to migration assay. After 2 days of activation, 0.5 × 106 PBMCs were added in the upper insert of the migration plates whereas 0.6 ml of supernatant from HIV-1 exposed or non-exposed FDCs were added in the lower insert. After 4 h at 37 °C, the inserts were removed, the cells collected from the lower wells and counted in a hemacytometer. Thereafter, the migrated cells were fixed with 2 % PFA and the frequency of CD19 and CD3 positive cells among migrated cells was evaluated.
The monoclonal antibodies (mAbs) used to identify and characterize the PBMCs in the migration assay are PE anti-CD69, FITC and PE anti-CD19, APC and FITC anti-CD3, PE and FITC isotype antibodies all purchased from BD Bioscience.
The activation status of the cells was then investigated by measuring CD69 expression on CD19+ and CD3+ PBMCs by Flow cytometry prior to migration assay. After 2 days of activation, 0.5 × 106 PBMCs were added in the upper insert of the migration plates whereas 0.6 ml of supernatant from HIV-1 exposed or non-exposed FDCs were added in the lower insert. After 4 h at 37 °C, the inserts were removed, the cells collected from the lower wells and counted in a hemacytometer. Thereafter, the migrated cells were fixed with 2 % PFA and the frequency of CD19 and CD3 positive cells among migrated cells was evaluated.
The monoclonal antibodies (mAbs) used to identify and characterize the PBMCs in the migration assay are PE anti-CD69, FITC and PE anti-CD19, APC and FITC anti-CD3, PE and FITC isotype antibodies all purchased from BD Bioscience.
3
Antibodies and Inhibitors for Signaling Pathways
4
BTK Activation Signaling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mino cells were treated
with indicated concentration of the compounds (3a –3g and 3j ). The cells were then incubated with
10 μg/mL anti-human IgM (Jackson ImmunoResearch, 109-006-129)
for 10 min at 37 °C and harvested. The cell pellets were subjected
to immunoblotting, and Western blots were performed for p-BTK, BTK,
and β-actin.
with indicated concentration of the compounds (
10 μg/mL anti-human IgM (Jackson ImmunoResearch, 109-006-129)
for 10 min at 37 °C and harvested. The cell pellets were subjected
to immunoblotting, and Western blots were performed for p-BTK, BTK,
and β-actin.
5
Quantification of PLCγ2 Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ramos cells (ATCC, Manassas, VA) were treated with titrating concentrations of BIIB091 for 30 min at 37°C in duplicates and subsequently stimulated for 5 min with anti‐human IgM (1 µg mL−1; Jackson ImmunoResearch, West Grove, PA). After centrifugation and removal of the supernatant, the cell pellets were lysed with gentle shaking in cold lysis buffer (Promega, Madison, WI) containing protease (Complete Mini; Roche, Basel, Switzerland) and phosphatase (PhosSTOP™, Roche); and phosphatase inhibition cocktail 2 and 3 (Sigma‐Aldrich) inhibitors. Cell lysates (diluted) were added and incubated for approximately 12 h at 4°C in MSD plate coated with anti‐PLCγ2 antibody (diluted 1:100, B‐10; Santa Cruz Biotechnology, Dallas, TX). Phosphorylated PLCγ2 was detected with rabbit polyclonal anti‐phospho tyrosine 1217 of PLCγ2 (diluted 1:1000; Cell Signaling Technologies) and Sulfo‐Tag goat anti‐rabbit antibody (diluted 1:500; MSD). Levels of phosphorylated PLCγ2 were detected as ECL signal on a Sector S600 (MSD) after the addition of 1× MSD Read Buffer.
6
Quantifying Phosphorylated Akt1 in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phospho-Akt1 (S473) sandwich ELISA antibody kit (Cell Signaling Technology, cat. no. 7143) was utilized to analyze pAKT signal in cells as described previously by Winkler et al. (11 (link)) (for data see Table 2 ). Briefly, SKOV3 and 786.0 cells were seeded into 96-well cell culture-graded plates at a density of two million per 200 µl culture media per well. Raji and Raw264.7 were seeded at the same density in FBS-free media. After overnight incubation at 5% CO2 and 37°C, the cells were treated with inhibitor for 30 min. Raji cells were stimulated with 10 µg/mL anti-human IgM (Jackson ImmunoResearch) for 30 min and Raw264.7 cells with 25 nM C5a (RnD Systems) for 3 min in the presence of inhibitor. SKOV3 and 786.0 cells were not stimulated. Medium was then aspirated and 50 µL/well of ice-cold lysis buffer was added. pAKT level was determined according to the manufacturer’s instruction.
7
CLL Cell Viability Assay with NLC and Anti-IgM
8
Lipid-specific Antibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect IgM and IgG antibodies to lipids we used a method published previously (25 (link)) with minimal modifications (described below). We incubated the wells with one of the following lipids: L-α-phosphatidylcholine (PC), 3-sn-phosphatidylethanolamine (PE), L-α-phosphatidylinositol (PI), 3-sn-phosphatidyl-L-serine (PS), N-Acyl-4-sphingenyl-1-O-phosphorylcholine (SM), 3-O-suphohexylceramide (SUL) and diphosphatidylglycerol, or cardiolipin (CL) (Sigma-Aldrich, St. Louis, MO, USA). Samples were diluted 1/100 in blocking solution and added to the wells in triplicate. We detected the presence of IgM or IgG to lipids in serum samples using the secondary antibodies anti human IgM (Jackson ImmunoResearch) or anti human IgG (Jackson ImmunoResearch), respectively. Positive sera were defined when the optic density (OD) was higher than the third quartile 3 (Q3) of the control group.
9
Anti-IgM Induced BTK Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the indicated cell
treatment, BTK phosphorylation was induced with 10 μg/mL anti-human
IgM (Jackson ImmunoResearch, 109-006-129) for 10 min at 37 °C.
The cells were harvested, and immunoblots of phospho-BTK, total-BTK,
and β-actin were performed.
treatment, BTK phosphorylation was induced with 10 μg/mL anti-human
IgM (Jackson ImmunoResearch, 109-006-129) for 10 min at 37 °C.
The cells were harvested, and immunoblots of phospho-BTK, total-BTK,
and β-actin were performed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!