Reversible fixing reagents and methods of use thereof

Patent metadata

Field	Value
Publication number	US-12566113-B2
Application number	US-202217845331-A
Country	US
Kind code	B2
Filing date	Jun 21, 2022
Priority date	Dec 23, 2019
Publication date	Mar 3, 2026
Grant date	Mar 3, 2026

Abstract

The present disclosure relates to compositions and methods for reversible fixation of biological samples using fixation reagents that form bis-carbamate crosslinks between amine-bearing moieties in biomolecules.

First claim

What is claimed is:

1 . A method for preparing a biological sample comprising contacting the sample with a first fixing reagent composition comprising:
a compound of formula (I)

wherein, “Linker” is a linear alkane moiety of 2-24 carbons; a linear alkane moiety of 2-24 carbons comprising a disulfide bond; or a linear alkane moiety of 2-24 carbons substituted with —OR, wherein R is selected from —H, —O(CO)—CH 3 , and —O(CO)— imidazole; and m is 1 to 12; and
a carrier.

2 . The method of claim 1 , wherein the compound of formula (I) is a compound of formula (II)

wherein n is 1 to 13.

3 . The method of claim 2 , wherein the compound of formula (II) is selected from compounds (2a)-(2k)

4 . The method of claim 1 , wherein the compound of formula (I) is a compound of formula (IV)

wherein m is 1 to 11, and n is 1 to 11.

5 . The method of claim 4 , wherein the compound of formula (IV) is selected from compounds (4a)-(4d)

6 . The method of claim 1 , wherein the compound of formula (I) is compound of formula (V)

wherein R is selected from —H, —O(CO)—CH 3 , and —O(CO)— imidazole.

7 . The method of claim 6 , wherein the compound of formula (V) is selected from compounds (5a)-(5c)

8 . The method of claim 1 , wherein the first fixing reagent composition comprises:
(a) the compound of formula (I) at a concentration of 5 mM to 50 mM; and (b) DMSO at a concentration of 2.5% to 5%.

9 . The method of claim 1 , wherein the method further comprises contacting the sample with a second fixing reagent, wherein the second fixing reagent comprises:
i) paraformaldehyde; ii) a compound of formula (III)

wherein n is 1 to 12; or
iii) a compound of formula (V)

wherein R is selected from —H, —O(CO)—CH 3 , and —O(CO)— imidazole.

10 . The method of claim 1 , wherein the method further comprises contacting the sample with a lysis agent and/or an unfixing agent, wherein the lysis agent comprises a compound capable of cleaving a carbamate bond or a compound capable of cleaving a disulfide bond and the unfixing agent comprises a compound capable of reversing paraformaldehyde fixation, wherein:
the compound capable of cleaving a carbamate bond is DETA, EDA, hydrazine monohydrate, a carboxyesterase, or a combination thereof; the compound capable of cleaving a disulfide bond is DTT; and the compound capable of reversing paraformaldehyde fixation is selected from any one of compounds (7a)-(7o)

11 . The method of claim 1 , wherein the biological sample is a tissue sample.

12 . The method of claim 5 , wherein the compound of formula (IV) is compound (4a).

Assignees

• 10X Genomics Inc

Inventors

• Delaney Joshua
• Gohil Shalini
• Lowe Adam
• Luo Yi
• Walter Dagmar

Classifications

• G01N2001/305

  Fixative compositions · CPC title

• C07D403/12

  linked by a chain containing hetero atoms as chain links · CPC title

• G01N1/30 Primary

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title