Fe-citrate
If you used 10 mM Fe-citrate, instead of 89.4 mM, the calculations would be easier.
The 89.4 value is because Tobias Baskin’s lab makes a 89.4 µM Fe-citrate medium, and it’s easy to mix up a batch from the 1000x stock solution.
For convenience, all staining, incubation, etc. will be done in HBS.
Each group does 4 coverslips, all of the same cell type. Some will use LLCPk, others will use 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 for coverslips 1-3
- 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
- high-Fe-HBS for coverslip 4
- 2 mL aliquots COLD
- 2 mL aliquots WARM
- need some to make high-Fe-HBS-BSA for transferrin
- Make 50 mL
| 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
- High-Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL High-Fe-HBS
- Transferrin in Fe-HBS-BSA to treat coverslips 1-3.
- 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 |
- Transferrin in high-Fe-HBS-BSA
- Each group does 1 50 µL treatment.
- Make 0.44 mL: 7 µL AF-488-Tf + 0.433 mL high-Fe-HBS-BSA
- Aliquot 55 µL
| # aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
|---|---|---|---|---|---|---|---|---|---|
| vi (Tf) | 0.88 | 1.76 | 2.64 | 3.52 | 4.4 | 5.28 | 6.16 | 7.04 | µL |
| HBS | 54.1 | 108.2 | 162.4 | 216.5 | 270.6 | 324.7 | 378.8 | 433.0 | µL |
| vf | 55 | 110 | 165 | 220 | 275 | 330 | 385 | 440 | µL |
- non-CO2 medium ICE COLD USE Fe-HBS – change manual !!!
- 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 hoodIngredientunits10152025405060paraformaldehydeg0.37.55520.9251.481.852.22watermL913.51822.5364557.7810X HBSmL11.522.5456
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood