Antibodies were obtained from the following suppliers: anti-human CD3 (BD Biosciences, 555335), anti-human CD8 (BD Biosciences 555366), anti-human CD107a (BD Biosciences 555801), anti-human CD137 (BD Biosciences 555956). Cell surface expression of ErbB2 was detected by biotylated anti-ErbB2 Affibody (Abcam, ab31890), and EGFR by FITC conjugated anti-EGFR affibody (Abcam, ab81872). EGFR, ErbB2 and CD19 specific CAR expression were detected by biotin-labeled polyclonal anti-human F(ab)2 antibody for (EGFR CAR) or anti-mouse F(ab)2 antibody (for ErbB2 and CD19 CARs)(Jackson Immunoresearch). Samples were then stained with PE-conjugated anti-human IgG Fc Ab (eBioscience, 12-4998-82) or phycoerythrin-labeled streptavidin (eBioscience, 17-4317-82). Flow cytometry acquisition was performed on either a BD FacsCalibur or Accuri C6 Cytometer (BD Biosciences). Analysis was performed using FlowJo software (Treestar).
Anti mouse f ab 2 antibody
Manufactured by Jackson ImmunoResearch
Sourced in Panama
Anti-mouse F(ab')2 antibody is a secondary antibody that specifically recognizes the F(ab')2 region of mouse primary antibodies. It is commonly used in immunoassays and other applications where detection of mouse antibodies is required.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Anti human cd3, by BD (2 mentions)
Anti human cd107a, by BD (2 mentions)
Anti human cd137, by BD (2 mentions)
Accuri c6 cytometer, by BD (2 mentions)
Anti human cd8, by BD (2 mentions)
Phycoerythrin labeled streptavidin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Cd23 coated microbeads, by Miltenyi Biotec (2 mentions)
Leaf purified anti cd40 antibody hm40 3, by BioLegend (2 mentions)
Ab81872, by Abcam (1 mentions)
Viability dye 7 aad, by BD (1 mentions)
Monoclonal antibodies specific, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Tim 3, by BD (1 mentions)
Dnam 1, by BD (1 mentions)
6 protocols using anti mouse f ab 2 antibody
1
Multiparametric Flow Cytometry Characterization
2
T Cell Surface Marker Detection
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Antibodies were obtained as follows: anti-human CD3 (BD Biosciences, 555335), anti-human CD8 (BD Biosciences, 555366), anti-human CD107a (BD Biosciences, 555801), and anti-human CD137 (BD Biosciences, 555956). The antibodies were incubated with T cells at 4°C for 25 min and washed twice (PBS with 2% FBS). Mesothelin CAR, ErbB2 CAR, CD19 CAR, and OKT3-28BB expression were detected by biotin-labeled polyclonal anti-mouse F(ab)2 antibody (Jackson Immunoresearch). Samples were then stained with phycoerythrin-labeled streptavidin (eBioscience, 17-4317-82). Flow cytometry acquisition was performed on either a BD FACSCalibur or Accuri C6 Cytometer (BD Biosciences). Analysis was performed using FlowJo software (Treestar).
3
Purification and Culture of Mouse Splenic B Cells
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Naive splenic B cells were purified by positive selection using CD23 coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by magnetic selection of labeled cells (B cell purity > 95%). All primary mouse B cells were cultured in RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermoscientific, Logan, Utah), 2 mM L-glutamine, penicillin (50 U/ml), streptomycin (50 U/ml), pyruvate (1 mM), MEM NEAA (100 uM), 2-ME (55 uM), HEPES (10 mM). For experiments in which B cells were stimulated in Ca2+ free media, 0.5 mM EGTA was added to complete RPMI media.-For in vitro stimulation assays, BCR was engaged using soluble 10 mg/mL anti-mouse F(ab′)2 antibody (Jackson ImmunoResearch, West Grove, PA) unless indicated otherwise. For CD40 engagement, LEAF purified anti-CD40 antibody (HM40–3, BioLegend, San Diego, CA) was used at a final concentration of 2 ug/mL. CpG (ODN1826: 5′-TCC ATG ACG TTC CTG ACG TT-3′) was synthesized and high-performance liquid chromatography purified by Integrated DNA Technologies (Coralville, IA). CpG was used at a final concentration of 1 µM. BLyS (R&D Systems, Minneapolis, MN) was used at final concentration of 100 ng/mL.
4
Flow Cytometry Analysis of UCART19 Cells
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Example 3
Commercially available anti mouse F(ab′)2 antibody from Jackson Immunoresearch and antibodies A8 and E11 antibodies described in Examples 1 and 2, were tested at four UCART19 concentrations (blank, 100,000 cells per mL in blood, 1 million cells per mL in blood and 10 million cells per mL in PBS). Exemplary flow cytometry plots are shown in
The A8 antibody detects the UCART19 positive cells in the absence of aspecific binding. The E11 antibody shows slight aspecific binding. The A8 and E11 antibodies give a strong PE positive signal allowing clear discrimination between UCART19 positive and negative cells. A ten-fold increase in % CAR+ cells is seen between tube B and C both for detection with the A8 and E11 antibody. For tube D, a 20-fold increase is seen rather than the expected 10-fold increase.
5
Purification and Culture of Mouse Splenic B Cells
6
Phenotypic Characterization of NK-92 Cells
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For flow cytometry, NK-92 cells were stained with monoclonal antibodies specific for CD56, CD3, CD45, CD28, CD16, CD11a, CD2, NKG2D, NKp30, NKp46, DNAM-1, CD95 (BD Bioscience) for 30 minutes on ice. CAR expression on the surface of NK-92 cells was detected with an anti-mouse F(ab’)2 antibody (Jackson Immunoresearch). For checkpoint molecules, antibodies specific for PD-1, LAG-3 and TIM-3 (BD Biosciences) were used. Dead cells were excluded using 7-AAD viability dye (BD Biosciences). Samples were acquired using an LSR-Fortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo 10 software (BD Biosciences).
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