Cells 2012

Cells 2012 kdorfman Tue, 07/03/2012 - 17:26

Lab 1: FL microscope

Lab 1: FL microscope kdorfman Mon, 07/30/2012 - 18:03

10 slides w/ DAPI

(1 per microscope + 2 extra)

Micrometers

Lab 2: Organelles

Lab 2: Organelles kdorfman Mon, 07/30/2012 - 18:26

5 or 6 slides each:

  • Rhodamine Phalloidin "1G"
  • Stained for rat anti-tubulin + goat anti-rat FITC (or GFP-tubulin cells) "2B"
  • Acridine orange stained for nucleoli "3G"
  • Stained with with mouse anti-Golgi + goat anti-mouse 568 "4G" (formaldehyde fixed)
  • Stained with rabbit anti-ZO1 + goat anti-rabbit Cy (cell-junction) "5G"

Lab 3: live cells; staining

Lab 3: live cells; staining kdorfman Mon, 07/30/2012 - 18:31

20 fixed coverslips for phalloidin staining

3 MatTeks per group for mitotracker and ?

aliquots of

  • phalloidin
  • mitotracker
  • non-CO2 medium

Lab 4 - discussion

Lab 4 - discussion kdorfman Mon, 07/30/2012 - 20:15

Finish looking at slides, discuss cell division

Lab 5: mitosis

Lab 5: mitosis kdorfman Mon, 07/30/2012 - 20:18

10 slides DAPI + tubulin

2 MatTeks per group each:

  • LLCPK with GFP tubulin
  • LLCPK parentals

NUC-Blue for staining chromosomes in live cells (Invitrogen R37605 or Fisher NC0291762), 1 bottle per station

  • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Non-CO2 medium

Lab 6: Mitosis

Lab 6: Mitosis kdorfman Mon, 07/30/2012 - 20:45

4 MatTeks per group of LL-GFP-tubulin

Double-strength inhibitors, made up in non-CO2 medium:

  • STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

  • need 1 mL aliquots 10 µM in non-CO2 medium for students

  • Make 100 mM stock:

    • 1 g in bottle.
    • Mix with 27.5 mL DMSO
    • heat to 65C (~1 hr), vortex
    • if necessary, filter sterilize to remove insoluble particles.
    • save as 1 mL aliquots. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)
  • Make 1 mM stock from the 100 mM stock

    • 10 µL 1mM stock
    • 990 µL DMSO
    • vortex
    • it will crystallize in the refrigerator. Warm and vortex to redissolve it.
  • Make 17 mL 10 µM in medium for students

    • 170 µL 1 mM stock
    • medium to 17 mL
    • aliquot
  • Make 10 µL aliquots of 1 mM stock

    • label says to add 990 µL medium to make 10 µM working solution

from Alyssa:madke STLC in DMSO and that she heated to 42C, and vortexed. She says it went from clear to yellow/brown. we use the reagent at micromolar - 1-10 µM. Her stock solution was 1mM.

students will be adding 1 mL of inhibitor to 1 mL medium in dish; the ready-to-use 1 mL (in medium) should be at least 2µM

  • Taxol (20 µM final)
  • soluble in DMSO, not water.
    • students pick either Taxol or nocodazole, so make only 10 each
    • each tube has 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)
    • add 490 µL to make 200µM in two tubes!
    • mix with medium in 15 mL conical to make 10 mL at 20 µM
  • Nocodazole (3.3 µM final)

    • each tube has 3 µL 33mM
    • add 97 µL to tube to make 100 µL of 1 mM
    • 33 µL 1mM + 10 mL medium = 3.3 µM
  • Nuc Blue from Invitrogen (or Fisher NC0291762), 1 bottle per station

    • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Lab 7: Cytokinesis

Lab 7: Cytokinesis kdorfman Mon, 07/30/2012 - 20:58

Myosin & tubulin expressing cells

2 MatTeks each per group

For ML-7, the final conc should be 75uM, so the 2X needs to be 150uM. For the Jasplak: the final conc should be 7uM so the 2X needs to be 14uM (but if 15 is easier, go with it). we use latrunculin at 5uM, and I think cytoD should be similar (I think I previously gave a range for cytoD of 1-10uM). Let's go with 5, so the 2X would be 10uM.

Inhibitors in non-CO2 medium (1 mL aliquots) each group does one (so make 5 of each):

  • cytochalasin D Sigma C2618 or C8273

    • tubes have 40 µL 2.5 mM
    • label says add 460 µL to make 500 µL of 200 µM
    • need 10 µM (2x final working concentration)
    • 20 µL 2.5 mM
    • 5 mL non0CO2 medium
    • gives 5 1 mL aliquots at 10 µM
  • Jasplakinolide – Santa Cruz biochemical, product sc202191 F

    • inducer of actin polymerization & stabilization
    • F-actin probe
    • MW = ~710
    • powder is stable frozen over a year
    • stock is stable 3-4 mo at -20C
    • Make 1 mM stock with 50 µg in 70 µL DMSO
    • Add 5 mL non-CO2 medium to make ~14 µM
    • 50 nM to 5 μM working range
    • minutes to hours incubation time
  • ML-7 Sigma, 12764.

    • soluble 10mg/mL in 50% EtOH
    • MW = 452.74
    • 5 mg in bottle
    • add 1100 µL to make 10 mM solution
    • used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
    • 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
    • make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM

Lab 8: projects

Lab 8: projects kdorfman Mon, 07/30/2012 - 21:04

Students limited to 3 MatTeks of one of the following:

  • GFP tubulin
  • tubulin-myosin cells

Plus their choice of inhibitors:

  • latrunculin
  • taxol
  • nocodazole
  • jasplakinolide
  • ML-7
  • STLC

Lab 9: Reports

Lab 9: Reports kdorfman Mon, 07/30/2012 - 21:08

Report on mitosis experiments

RNA

RNA kdorfman Tue, 07/03/2012 - 17:40

Acridine orange

Acridine orange kdorfman Fri, 08/03/2012 - 21:01

Sigma A8097-10 mL

10 mg/mL = 33mM

MW = 301.81

Use at ~20µM

  • 1.2 µL added to 2 mL in dish

  • incubate 15 min at 37

  • change medium

  • incubate 15 min at 37

  • Replace medium with PBS

  • observe with

    • B excite - G emission: dsDNA
    • G excite - R emission: RNA, ssDNA

Got good results initially, but within minutes, cells started to ball up and pull off the substrate. Will try new PBS first, then a concentration gradient of acridine orange.

Old PBS produced fast shrinking and balling up. Newer PBS was less bad. Now try with non-CO2 medium. Does it interfere with fluorescence?

No cell shrinkage in non-CO2 medium. At short exposure times, there is a background glow, but picking the right exposure takes care of that.

This is time sensitive. The red emission (which should be RNA & ssDNA) gradually fades, or overlies the green emmision (which should be ds DNA)

See attached images, taken in order 1, 2, 3. #3 is at about 15 minutes. 1 & 2 are 100x, 3 is 200x.

SYTO RNASelec

SYTO RNASelec kdorfman Fri, 08/03/2012 - 20:59

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

  • Cells on coverslip
  • Warm 500 nM labeling solution to 37C
  • Incubate at 37C 20 min
  • Rinse twice in PBS or medium
  • Add warm medium, let cells rest 5 min
  • Fix in chilled methanol 10 min at -20C
  • Several washes in PBS

Fixed cells

  • Remove coverslip from medium
  • Fix in chilled methanol 10 min at -20C
  • Remove methanol, let slip sit in PBS 5 min
  • Apply labeling solution 20 min RT
  • Wash 5 min in PBS
  • Mount coverslip