STANDARD OPERATING PROCEDURE Page 1 of 3

STANDARD OPERATING PROCEDURE

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CYTO.SAMPLE.001.00

TITLE: SECTION: SOP NUMBER: REVISION: EFFECTIVE DATE: ORIGINATED BY: REASON FOR REVISION: APPROVAL DATE:

LYSING RED BLOOD CELLS FROM A TISSUE SAMPLE CYTOMETRY CYTO.SAMPLE.001 00 WUNDERLICH, JOSHUA NEW SOP

REVIEWED BY (AA): NAME

SIGNATURE

DATE

NAME

SIGNATURE

DATE

NAME

SIGNATURE

DATE

REVIEWED BY:

APPROVED BY:

1000 East 50th Street, Kansas City, Missouri 64110 Telephone: (816) 926-4108, Fax: (816) 926-4671 www.stowers-institute.org

STANDARD OPERATING PROCEDURE

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CYTO.SAMPLE.001.00

Scope:

During flow cytometric analysis of a tissue sample, red blood cells tend to interfere with lymphocytes as well as overwhelm the entire white blood cell (WBC) population in number; in order to create an assay that exclusively evaluates lymphocytes and WBC, red blood cells in a sample are removed from solution by lysis. Any Facility member or researcher working in collaboration with the facility that has received proper technical lab training can perform this task

Purpose:

Describe a consistent, reliable method for fully lysing red blood cells from a sample with minimal damage done to the remaining WBC specimen.

Definitions:

None.

Responsibility:

Any person processing vertebrate hematopoietic tissue for the purpose of cytometric analysis/sorting is expected to follow this procedure.

Equipment:

Refrigerated Centrifuge

Procedure:

I.

II.

III. IV.

V.

VI. VII. VIII.

If 10x Hemolytic Stock Buffer (a.k.a., 3.1 M Ammonium chloride RBC lyse solution) was prepared more than three months prior to the date of the experiment, prepare 500 ml of new stock buffer by the following method: a. Mix the following reagents: 300 ml i. Sterile H2O ii. NH4Cl 83 g iii. NaHCO3 10 g iv. EDTA 370 mg b. QS solution to 500 ml with sterile H2O. c. Shake solution for 15 minutes, or until no precipitate remains. Prepare fresh 1x Hemolytic Buffer by mixing nine parts sterile H2O with one part 10x Buffer. Prepare such a volume to ensure 20 ml of 1x Buffer per sample. Warm prepared 1x Buffer to 37°C in a water bath. Immediately upon receipt of the samples, concentrate them by centrifuging at 500g for 5 min in 50 ml Polypropylene conical tubes (BD Item No. 352098) and decant the supernatant. Resuspend pellet by vortexing for three seconds and add 15 ml 1x Hemolytic Buffer to each sample and repeatedly invert for three minutes. After three minutes, QS all samples to 50 ml using a solution of PBS that includes 2% FBS and 0.1% NaAz. Invert samples for ~15 seconds and centrifuge at 500 g for 5 minutes. Resuspend samples in ~1 ml of the aforementioned PBS/FBS/NaAz solution (by vortexing), and force samples through 70 um filters, 1000 East 50th Street, Kansas City, Missouri 64110 Telephone: (816) 926-4108, Fax: (816) 926-4671 www.stowers-institute.org

STANDARD OPERATING PROCEDURE

Page 3 of 3

CYTO.SAMPLE.001.00

IX.

followed by ~1 ml more of the PBS solution, into 5 ml Polypropylene round bottom tubes (BD Part No. 352063). Concentrate samples as previously described, decant and resuspend in 1 ml of the PBS solution. Begin preparation before sample arrives.

Is 10x buffer more than 3 months old?

Y Prepare new 10x buffer.

N Prepare 1x buffer

Receive Samples

Concentrate Samples

Lyse Samples and wash

Filter Samples and wash

Resuspend in 1 ml PBS+FBS to count

References:

None

Appendices:

None.

1000 East 50th Street, Kansas City, Missouri 64110 Telephone: (816) 926-4108, Fax: (816) 926-4671 www.stowers-institute.org