Cellular Immunology Module
Lab 1
Lecture: Introduction
Film: Laboratory Safety.
Lab 2
Lecture: Cell counting and cell viability.
Films: Aseptic Technique I, Aseptic Technique II.
Laboratory:
Cell Count and Cell Viability: Parts A-G.
Lab 3
Lecture: Monoclonal antibody production and cell fusion experiment.
Laboratory:
Cell Fusion for Production of Hybridomas: Parts A and B, Steps 1-13. Perform fusion and plate hybridomas into HAT medium in 96-well plates.
Lab 4
Lecture: Lac Z T cell activation assay.
Laboratory:
LacZ T cell Activation Assay: Part A. Set-up cultures for T cell activation.
Protein Assay
Lab 5
Laboratory: Lac Z T cell Activation Assay: Part B. Add CPRG substrate.
Lecture: ELISA
Laboratory:
Enzyme-Linked Immunosorbent Assay (ELISA) for Antibody:
DAY 1 steps 1-2: Coat plates.
Cell Fusion for Production of Hybridomas: Part B, step 14.
Observe and feed cultures.
Lab 6
Laboratory: ELISA for Antibody: DAY 2. Block plate before lecture.
Lecture: Cloning
Laboratory: ELISA for Antibody: DAY 2.
Cell Fusion for Production of Hybridomas: Part C. Transfer supernates to corresponding wells of transfer plate and feed cultures on hybridoma master plate.
LacZ T cell Activation Assay: Analyze data in computer room.
Lab 7
Laboratory: ELISA for Antibody: DAY 3.
Complete ELISA assay on hybridoma supernates and record data.
Check results of ELISA assay with Instructors before selecting well from hybridoma master plate to use for cloning.
Limiting Dilution Cloning: Parts A-C. Clone best antibody-producing hybridoma from hybridoma master plate.
Cell Fusion for Production of Hybridomas: .
Record growth of hybridoma cultures from master plate.
Lab 8
Data Summary on LacZ Assay for T Cell Activation due.
Lecture: Immunoprecipitation.
Laboratory: Immunoprecipitation: Part A: Label mouse hybridoma cells with biotin, prepare cell lysate. Part B, steps 1-2: Clear lysate.
Lab 9
Lecture: SDS PAGE and Western Blot
Laboratory:
Immunoprecipitation: Part B, steps 3-10: Precipitate mouse IgG from cell lysates with goat anti-mouse IgG-agarose and prepare samples for electrophoresis.
Limiting Dilution Cloning: Part D.Observe both cloning plates and potentially feed just one cloning plate.
Lab 10
Lecture: SDS-PAGE and Western Blot: logistics
Laboratory: SDS-PAGE: Parts A -C. Prepare samples, run gels.
Western Blot: Parts A and B. Perform electrophoretic transfer of proteins from slab gel to nylon membrane.
Lab 11
Laboratory: Western Blot:
Part C. Add antibodies to nylon membranes before lecture.
Lecture: Science Writing Lecture.
Laboratory: Western Blot: Parts C and D. Develop nylon membranes with labeled antibodies and plot MW standard curve.
ELISA for Antibody. DAY 1. Steps 1-2; Coat plates with antigen.
Antigen Capture ELISA: DAY 1. Steps 1-2: Coat plates with antibody.
Lab 12
Laboratory Report on Immunoprecipitation due
Lecture: Assay of cloning plates and evaluation of specificity of monoclonal antibodies. Criteria for completion of cloning.
Laboratory: ELISA for Antibody. DAY 2. Block plates, add samples.
Antigen Capture ELISA: DAY 2. Block plates, add samples.
Limiting Dilution Cloning: Record growth and transfer culture supernates to antigen-coated plate for assay.
Lab 13
Laboratory: Add secondary antibody before lecture.
Lecture: Flow cytometry
Laboratory: Finish ELISA for Antibody. DAY 3. Steps 9-16;
Antigen Capture ELISA: DAY 3. Steps 7-15.
Lab 14
Laboratory Report on Production of Monoclonal Antibodies Due
Laboratory: Flow cytometry: Stain cells and analyze on flow cytometer.
