W 9/04

Intro to Equipment

W 9/09

Imaging mitosis with fixed cells. Image Spatial Quantification

W 9/11

Image Formation in the Microscope

M 9/16

Numerical Aperture

W 9/18

Resolution

M 9/23

Fluorescence Microscopy

W 9/25

Identification of Cellular Organelles

M 9/30

Labeling Cellular Structures

Cells

• paraglut fixed LL's for students to stain for tubulin (make extra!)

• paraglut fixed LL's for students to stain for phalloidin

• paraglut fixed 3t3's for students to stain for phalloidin

• live LL's to fix in paraglut (2014: treat first with mitotracker, then fix in formaldehyde no detergent)

Solutions

• PBS to rinse coverslips

• paraglut

• formaldehyde

• PBS-Tw-Az

• 2% PBS-BSA to make up antibodies

Materials

• forceps

• 50 mL beakers (switch to holders and 100 mL beakers?)

• petri dishes for humid chambers

• filter paper for humid chambers

• mounting medium

• slides

• nail polish

• kimwipes

• parafilm

W 10/2

Imaging Living Cells Expressing GFP-Tagged Proteins

• LL-GFP-tubulin

• LL-GFP-actin

• LL nucleofected with plasmids

  • H2B

2014

• LL-EB1-GFP stable line

• LL-alpha tubulin=GFP stable line

** transfected**

• H2B m cherry

• alpha actininin (GFP?)

W 10/9

Motility in Control Cells

On MatTek dishes:

• 3t3

• B16

• LL-GFP actin

Non-CO2 medium

Tu 10/15 & W 10/16

Mechanism of Motility

Students check for effect of dose and time on actin filaments. Treat LL parentals with cytochalasin D, then stain actin with phalloidin.

• LL on coverslips

• non-CO2 medium

• Cytochalasin D

• paraglut

• PBS-Tw-Az

W 10/23

Receptor-mediated endocytosis

NO LL's

NO high iron

3 coverslips of 3t3 per group

Try Lysotracker (Invitrogen L-7528)

• 50 - 75 nM

• stock = 1 mM in DMSO

• 30 min - 2 hours incubation warm.

• Add lysotracker to cells at 1 pm.

  • Make 100 µM stock
  • 1 µL of 100 µM stock to each dish. Assume ~1.5 mL per dish

Omit the high-iron treatment

For convenience, all staining, incubation, etc. will be done in HBS.

Each group does 3 coverslips of 3t3.

• HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.

  • 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
  • 15 mL aliquots RT in hood (to rinse off fixative)
  • Need ~300 mL total

• Fe-HBS

  • 20 mL aliquots COLD to rinse off growth medium, then tf
  • 5 mL aliquots WARM (incubation after tf treatment)
  • need some to make Fe-HBS-BSA for transferrin
  • make 200 mL

plus water to final volume

• Fe-HBS-BSA 1 mg/mL to make transferrin solution

  • 5 mg BSA in 5 mL Fe-HBS

• Transferrin in Fe-HBS-BSA

  • Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
  • Each group does 3 50-µL treatments (need extra for pipetting error)
  • Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
  • Aliquot 165 µL
    if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

• HBS-paraformaldehyde 3.7%

  • Need 60 mL
  • Divided in 2 aliquots, one per fume hood

Old fashioned way:

heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood

OMIT HIGH IRON

plus water to final volume

M 10/28

Bulk-phase endocytosis

** 2 coverslips, 2 small diameter MatTek dishes 3t3

PBS for rinsing live cells WARM
PBS for rinsing fixed cells RT
Take from the supply of sterile aliquots

Growth media for incubation WARM
10 mL aliquots each

• DMEM for 3t3 cells
• non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)

TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)

Make 8.8 µL aliquots in 2 mL tubes.

TMR-dextran Working concentration = 0.05 mg/mL in medium
2 coverslips at 50 µL/coverslip (=~110 µL/group)

Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
Aliquot ~105 µL/group

TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
tmr as above

FITC-dextran Working concentration = 0.5 mg/mL in medium
(1mg/mL might be better, but that uses up a whole bottle.)
2 dishes at 100 µL/dish (=~220 µL/group)

Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex

Methylamine solution (1M)
Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
(Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)

Make 1 mL, aliquot ~120 µL

plus water to final volume

Fixative

• PBS-paraformaldehyde 3.7%
  
  • Need 70 mL
  • Divided in 2 aliquots, one per fume hood

heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood

OR http://wahoo.nsm.umass.edu/content/paraformaldehyde-fix

Cells

• 3t3 on coverslips - 2 per group
• 3t3 on MatTek dishes (small diameter)
• LLCPk GFP-alpha tubulin on coverslips 1 per group
• LL parentals on coverslips 2 per group

Reagents

HBS-BSA

TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)

TMR-dextran Working concentration = 0.05 mg/mL in medium

• 2 coverslips at 50 µL/coverslip (=~110 µL/group) + 2 live cells at 100 µL/mattek
  
• Aliquot 5.2 µL 10 mg/mL
• Label says to add 98.8 µL to make 0.5mg/mL working soloution

Lysotracker (Invitrogen L-7528 - 20 x 50 µL)

• 50 - 75 nM
• stock = 1 mM in DMSO
• aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
• 30 min - 2 hours incubation warm.

Transferrin in Fe-HBS-BSA

• Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
• Each group does 2 50-µL treatments (need extra for pipetting error)
• Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA

• Aliquot 165 µL
  

  if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

Cells

• 2 coverslips 3t3 per pair for TMR-dextran
• 2 LL-GFP-tubulin in MatTek dishes per pair for lysotracker & endosome movement
• 3t3 in MatTek for live studies

Reagents

• HBS-BSA (1 mg/mL). per pair ~7mL:

  • 6 mL for 3 rinses live cells
  • 1 mL for nocodazole
  • 0.1 mL for Transferrin

• Nocodazole 1 µM in HBS-BSA 1 mL per pair

  • aliquots are 3 µL 3mM in DMSO
  • add 97 µL to make 1mM stock
  • dilute 1:1000 in HBS-BSA (10 µL in 10 mL)

• Transferrin in Fe-HBS-BSA to label live cells 100 µL/pair

  • Stock is 5 mg/ml (=62.5 µM)
  • Working conc = 1 µM
  • 0.016 µL Tf/µL solution
  • 1 mL = 16 µL Tf + 984 µL Fe-HBS-BSA

• Lysotracker in non-CO2 medium 1mL/pair

  • stock is 1 mM
  • working solution is 50 - 75 nM
  • Dilute 7.5:100,000 = 0.75 µL/10 mL

• TMR-dextran (Invitrogen D3308, 10 mg) in non-CO2 medium

  • 100 µL to stain in dish
  • 100 µL to stain 2 coverslips
  • 10 mg/mL stock, in 8.8 ML aliquots
  • 0.05 mg/mL working concentration
  • aliquot ~210 µL per pair

• CO2 medium (to rinse and return to incubator)

• Non-CO2 medium (for imaging)

• PBS for rinsing

• 3.7% paraformaldehyde in PBS for fixation: 3 mL/pair

W 10/30

Transport of endosomes along microtubules

3 small-diameter MatTek dishes of LLCPk GFP tubulin

HBS-BSA 1mg/mL

• to mix transferrin ( 3 mL) and nocodazole (15 mL) in

• to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 8 groups = 144 mL. Aliquot 20 mL.

• make 250 mL

Fe-HBS-BSA
to mix transferrin in
Need 2.3 mL; make 3 mL

in Fe-HBS-BSA to final volume

MAKE ENOUGH OF EACH FOR EACH GROUP TO DO 3 TREATMENTS

Transferrin in Fe-HBS-BSA
Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
stock is 5 mg/mL (=62.5 µM).
Final concentration = 1 µM.

• Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
• Make 105 µL aliquots (21)
• Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA

if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.

Nocodazole
Acros 358240100, 10 mg
1µM in HBS-BSA
(Working concentration range is usually 100nM - 30 µM.)
Stock is 10 mg/mL (=33mM)
(Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)

Make enough for each group to do two 1 mL treatments.
Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL

• Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
• Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL

non-CO2 medium
to mix TMR-dextran in
to incubate cells in

TMR-dextran

in non-CO2 medium
Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.

Invitrogen D3308, 10 mg
Make 100 mg/mL stock (add 100µL to the bottle)
1 µL aliquots

TMR-dextran Working concentration = 0.05 mg/mL in medium
100 µL per treatment
Aliquot 105 µL, make enough for each group to do 3

Make 10 mg/mL dilution to make student aliquots
1 µL 100 mg/mL stock + 9µL medium

M 11/4

Cellular responses to external stimuli

3t3 on large diameter MatTek dishes

4 dishes per group plus extra = 36

Plate 2 days before class.

W 11/6

Release of internal calcium stores

3t3 on large-diameter MatTek dishes

4 dishes per group (plus extra) ~36

starting W 11/13

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