RIME CELL FIXATION PROTOCOL
1. Fix 5 x 107 to 108 cells for each cell population to be tested. To fix, add 1/10 volume of freshlyprepared Formaldehyde Solution* (see Reagents below) to the existing media. Do NOT remove
existing media. For example, to a flask containing 10 ml of media, add 1 ml of Formaldehyde
Solution. Cap and agitate for 8 minutes at room temperature.
2. Stop the fixation by adding 1/20 volume Glycine Solution* to the existing media in each container.
For example, if the flask from Step 1 now contains 11 ml, add 0.55 ml 2.5 M glycine. Let sit at room
temperature for 5 minutes. After the glycine incubation, if the cells are adherent, scrape themthoroughly from the culture surface.
3. Wash cells by transferring contents of each container to a conical tube (15 ml or 50 ml tube,depending on the volume). Keep samples on ice for the remainder of the procedure. Centrifuge
tubes at 800 x g in a refrigerated centrifuge for 10 minutes to pellet the cells. Remove thesupernatant and re-suspend cells in 10 ml chilled PBS-Igepal* per tube by pipetting up and down. Ifcells from any one population are contained in multiple centrifuge tubes, combine them at this step.
4. Centrifuge tubes again as before to pellet the cells. Remove the supernatant, then add 10 ml chilled
PBS-Igepal* to each tube. Add 100 µl PMSF (100 mM in ethanol*; final concentration will be 1 mM)
to each tube and pipet up and down to re-suspend the cells.
5. Centrifuge tubes a third time to pellet the cells, and carefully remove supernatant completely fromcell pellets.
6. Snap-freeze cell pellets on dry ice and store at -80°C.

Final
concentration

Reagents*

1. Formaldehyde Solution (to be prepared fresh before use):
16% Methanol Free Formaldehyde(e.g. ThermoFisher #28908)
11%
5 M NaCl
0.1 M
0.5 M EDTA, pH 8.0
1 mM
1 M HEPES, pH 7.9
50 mM
H2 O
(Note: NaCl, EDTA, and HEPES should be molecular biology grade.)
2. Glycine Solution
Glycine, MW 75 (e.g. Sigma #G-7403)
H2 O

2.5 M

3. PBS-Igepal
PBS, pH 7.4 (e.g. ThermoFisher #10010023)
100% Igepal CA-630 (e.g. Sigma #I-8896)

~1X
0.5%

Per 10 ml

6.9 ml200 µl20 µl500 µl2.4 mlPer 10 ml
1.88 gto 10 ml
Per 100 ml
100 ml
0.5 ml4. PMSF (e.g. Sigma #P-7626)
Prepare at 100 mM in ethanol and store at -20°C.
(Note: PMSF Phenylmethanesulfonyl fluoride.)North America 877 222 9543
RIME Sample Prep

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

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Rev B_102023

RIME Tissue Preparation Protocol
If you are planning on submitting tissue samples for RIME services, freeze tissue according to oneof the protocols below. Tissue requirements for RIME services is a minimum of 800 mg.

Consumables-Cryogenic vial(s) or 2 ml low-bind microcentrifuge tube(s)Liquid Nitrogen
Dry ice

Protocol A: Liquid Nitrogen
1. Excise the tissue and place in a cryogenic vial or microcentrifuge tube
2. Immediately submerge tube in liquid nitrogen for 2 minutes
3. Store at -80oC

Protocol B: Dry Ice
1. Excise the tissue and place in cryogenic vial or microcentrifuge tube
2. Immediately place tube on dry ice for 15 minutes
3. Store at -80oC

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com
Rev B_102023