================ TC media recipes ================ N3 media -------- Used for neuronal differentiation during direct conversion. * Total volume: **500 mL** =============================== =========== =============== Component % Volume =============================== =========== =============== Glutamax (1%) 1% 5 mL N2 (100x stock) 100x stock 5 mL B27 (50x stock) 50x stock 10 mL Pen/Strep (1%) *(optional)* 1% 5 mL FBS *(optional)* 2% 10 mL DMEM F12 500 mL =============================== =========== =============== Add the following small molecules to small volumes immediatly before media addition, to a **final concentration of 10 ng/mL**: * BDNF, from 1,000x stock (10,000 ng/mL) * CNTF, from 1,000x stock (10,000 ng/mL) * GDNF, from 1,000x stock (10,000 ng/mL) * FGF, from **10,000x** stock (100,000 ng/mL) * RepSox, **if RR**, from 1000x stock (7.5mM) .. note:: 1. 200mM glutamine is the same as Glutamax 2. We do not usually use FBS or Pen/Strep 3. Wrap N3 in foil after making MEF media --------- Used for culturing MEFs and other fibroblasts. Total volume: **500 mL** ========================= ===== =============== Component % Volume ========================= ===== =============== FBS 10% 50 mL Pen/Strep *(optional)* 1% 5 mL DMEM add to 500mL ========================= ===== =============== Breast cancer 1 media --------------------- Use for culturing the **LM2**, **BT-20**, and **MDA-MB-468** cell lines. =============== =============== ================ Component Concentration Volume / 50 mL =============== =============== ================ DMEM + 10% FBS Base Use MEF media Anti-Anti 1% 500 uL =============== =============== ================ Breast cancer 2 media --------------------- Use for culturing the **HS 578T** and **BT-549** cell lines. =============== =============== ======================== Component Concentration Volume / 50 mL =============== =============== ======================== DMEM + 10% FBS Base Use MEF media PSG 1% 500 uL Insulin 10 ug/mL 125 uL of 4 mg/mL stock =============== =============== ======================== Breast cancer 3 media --------------------- Use for culturing the **MDA-MD-231** cell line. =============== =============== ======================== Component Concentration Volume / 50 mL =============== =============== ======================== DMEM + 10% FBS Base Use MEF media PSG 1% 500 uL =============== =============== ======================== Breast cancer 4 media --------------------- Use for culturing the **SUM159** cell line. =============== =============== ======================== Component Concentration Volume / 50 mL =============== =============== ======================== F12 Base 47 mL FBS 5% 2.5 mL Anti-anti 1% 500 uL Insulin 5 ug/mL 62.5 uL of 4 mg/mL stock Hydrocortisone 1 ug/mL EGF 20 ng/mL 1 uL of 1 mg/mL stock =============== =============== ======================== Epithelial cell 1 media ----------------------- Use for culturing the **NCI-H1299** human lung cancer cell line. =============== =============== Component Concentration =============== =============== RPMI + 10% FBS Base =============== =============== Epithelial cell 2 media ----------------------- Use for culturing the **SAOS-2** human osteosarcoma cell line. ================= =============== Component Concentration ================= =============== McCoy's + 15% FBS Base ================= =============== Glia media ---------- Used for culturing glia cells Total volume: **500 mL** ========================= ===== =============== Component % Volume ========================= ===== =============== Horse serum 10% 50 mL Glucose 20% 100 mL MEM 70% 350 mL ========================= ===== =============== .. _sortmedia: Sorting/Collection media ------------------------- Used for cell sorting. Use DMEM/F12 for flow sorting and DMEM/F12 + 10% FBS for collection to help make cells happier. It is possible to use whatever though because you will have to spin-down and resuspend in the correct media (i.e. N3) anyways. You will want to include Pen/Strep as it will greatly reduce the amount of contamination. Total volume: **50 mL** ================================= ===== =============== Component % Volume ================================= ===== =============== FBS *(for collection only)* 10% 5 mL DMEM/F12 89% 44.5 mL 100X Pen/Strep 1% 500 µL ================================= ===== =============== .. _MNdissociate: Motor neuron dissociation media ---------------------------------- Used for dissociating iMNs or primary motor neurons (embMN) harvested from spinal cords for plating/sorting. Add 50 µL/96-well, let sit in 37°C for ~15 min. Lightly tap plae to see if cells are dissociating. Total volume: **6 mL** (enough for 1x96-well) ========================= ========================= Component Volume ========================= ========================= Papain 1 vial (>= 100 U/vial) DNAse 1 vial (>= 1,000 U/vial) DMEM/F12 6 mL ========================= ========================= .. _freezingmedia: Freezing media -------------- ======================== =============== ======================== Component Volume (1 mL) Final Concentration ======================== =============== ======================== FBS (or DMEM/10% FBS) 900 µL 90% DMSO 100 µL 10% ======================== =============== ======================== - It is easy to keep a 4°C stock of (:ref:`80% FBS/20% DMSO `) then use 500 µL 80%/20% FBS/DMSO + 500 µL DMEM/FBS cell solution .. _HEPES: HEPES-buffered DMEM ------------------- For use during lentivirus or retrovirus production in HEK293T cells. We use HEPES Sigma-Aldrich (H3375). **1M HEPES stock solution** (filter sterilize with 0.22 µm filter after pH-ing) ============================== =============================== ============== Component Concentration Amount/50 mL ============================== =============================== ============== HEPES 1M 13.82 g DI H2O main solvent 50 mL NaOH to pH 7.0 ============================== =============================== ============== .. note:: When NBW did this on 7/10/23, I did 41.46 g HEPES + 150 mL water. The HEPES takes up a lot of volume so I would dissolve in 100 mL ELGA-filtered water, then check volume and increase to 150 mL. At the beginning, pH = 5.45 and I had to add 8.9 mL 4N NaOH to get to pH = 7.00. **HEPES-buffered DMEM** ============================== =============================== ============== ============== Component Concentration Amount/50 mL Bottle ============================== =============================== ============== ============== DMEM + 10% FBS main Component 48.75 mL 550 mL Sterile 1M HEPES 25 mM 1.25 mL 14.10 mL ============================== =============================== ============== ==============