1. Cytoskeleton Lab 2: The Effects of Cytochalasin B on the Actin Cytoskeleton & Cell Viability in Mouse Fibroblast Cells

2. Today: Two Assays 1.Actin staining Each group will assay two concentrations of cytochalasin B (CB) between 2 and 20 µM and will also do a control (no drug). Cells treated for 1 h. 2.Cell viability (flow cytometry) Each group will assay one of the two CB concentrations tested in the actin staining experiment (or a control with no drug). Will assay cells treated 1 h or 24 h with the assigned concentration of drug (24 h samples started yesterday). Data for the entire class will be pooled for your lab report Results.

3. Cytochalasin B Concentration Assignments: GroupActin StainingCell Viability 2, 10 µMControl 5, 20 µM5 µM 2, 10 µM10 µM 5, 20 µM20 µM

4. Before the Lab Intro: Obtain a 10-ml aliquot of culture medium containing 20 µM cytochalasin B (CB). Prepare 8 ml of medium containing your assigned CB concentration for the cell viability assay and 3 ml of medium containing your other assigned CB concentration for the actin staining assay. Obtain one 4-chamber growth slide containing cells; dump medium from chambers into waste beaker (in hood). Label frosted portion of slide with your initials. Add 1 ml fresh (drug-free) medium to 1 st 2 wells of each slide (closest to frosted part of slide); add 1 ml of high CB medium to next well and 1 ml of low CB medium to last well. Obtain one flask of cells diluted last week. Aspirate the culture medium and add 5 ml of medium containing your assigned CB concentration for the viability assay. Incubate both slide and flask 1h in tissue culture incubator (37°C, 5% CO 2 ).

5. The Actin Cytoskeleton Microfilaments formed from actin monomers Filaments have polarity: plus & minus (or “pointed” & “barbed”) ends Actin monomers add preferentially to plus/barbed ends

6. Roles of Actin Binding Proteins

7. Cytochalasin B Effects on Actin: Effects on Cells:

8. Molecular labeling techniques used for fluorescence microscopy 1.Direct: fluorescent molecule binds directly to target molecule. Ex: phalloidin conjugated to rhodamine (red fluorescent dye) binds specifically to F-actin, the filamentous form. 2.Indirect: fluorescent molecule binds to some other molecule used for recognition of the target molecule. Untagged primary antibody used to recognize target protein & dye- conjugated secondary antibody used to visualize.

9. Amanita phalloides “Angel of Death” mushroom Phalloidin locks F-actin subunits of actin together, stabilizing actin filaments. Phalloidin

10. Actin stained with Phalloidin conjugated to FITC or Rhodamine

11. Flow Cytometry Technique for measuring characteristics of cells (e.g. fluorescence, size, “granularity”) in a population on a cell-by-cell basis Cells flow through a capillary in “single file”, passing through a laser beam of a certain wavelength. The scattered light and fluorescence intensity are recorded for each cell. Fluorescence Activated Cell Sorting (FACS)—sophisticated flow cytometry

12. ViaCount Assay Determines % of living and dead cells in a culture All cells stain with a DNA dye (distinguishes from non- cell debris) & dead cells only stain w/ an exclusion dye (similar to Trypan Blue); two dyes emit fluorescence of different wavelengths Flow cytometer quantifies intensity of fluorescence of at the two wavelengths as each cell passes through the laser Sample Data:

13. Protocol Steps after Drug Incubation Actin StainingViability Assay PBS washes (4 x 3 min) Formaldehyde fixation (10 min) PBS washes (3 x 3 min) Cold acetone permeabilization (3 min) Rhodamine-phalloidin staining (20-25 min) PBS washes (4 x 3 min) Mounting medium, coverslip & seal Rinse cells w/ PBS Trypsinize (30 sec + 15 min) Resuspend in 5 ml fresh medium Place 200 µl each cell suspension in a regular microfuge tube Wait for entire class to be ready Mix cells with 2 µl 0.5X ViaCount reagent Do flow cytometry during rhodamine-phalloidin incubation PBS washes (3 x 3 min)

14. For Next Week (Week of April 16): Sign up for microscope time slot Bring research article for imaging method presentation to your microscope appt. for approval. For Week of April 30: 10-15 min group presentation on chosen imaging topic & its application in your chosen research paper For Week of May 7: Mini-lab report due (Title, Abstract, Results, References)