Reagents 2012
Reagents 2012 kdorfman Wed, 10/10/2012 - 21:16Omit 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
Solution | stock conc | final conc | 25 | 50 | 75 | 100 | 200 | mL |
---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 2.5 | 5 | 7.5 | 10 | 20 | mL |
Fe-citrate (mM) | 89.4 | 0.1 | 27.96 | 55.92 | 84 | 112 | 224 | µL |
plus water to final volume
- Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL Fe-HBS
Solution | stock conc | final conc | 10 | 15 | 20 | 25 | 30 | 50 | mL |
---|---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 1 | 1.5 | 2 | 2.5 | 3 | 5 | mL |
Fe-citrate (mM) | 89.4 | 2 | 223.7 | 335.6 | 448 | 559.2 | 671 | 1118.4 | µL |
plus water to final volume
- 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.
# aliquots | 3 | 6 | 9 | 12 | 15 | 18 | 21 | 24 | |
---|---|---|---|---|---|---|---|---|---|
vi (Tf) | 2.64 | 5.28 | 7.92 | 10.56 | 13.2 | 15.84 | 18.48 | 21.12 | µL |
HBS | 162.4 | 324.7 | 487.1 | 649.4 | 811.8 | 974.2 | 1136.5 | 1298.9 | µL |
vf | 165 | 330 | 495 | 660 | 825 | 990 | 1155 | 1320 | µL |
**ICE COLD Fe-HBS **
- 3 rinses per coverslip
- 15 mL aliquots in the refrigerator
- need over 100 mL
HBS-paraformaldehyde 3.7%
- Need 60 mL
- Divided in 2 aliquots, one per fume hood
Ingredient | units | 10 | 15 | 20 | 25 | 40 | 50 | 60 |
---|---|---|---|---|---|---|---|---|
paraformaldehyde | g | 0.37 | .555 | 2 | 0.925 | 1.48 | 1.85 | 2.22 |
water | mL | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 |
10X HBS | mL | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood