Enriching Rare Populations of Human CD34 Cells by FACS

Transcript

• 00:03Title of this training is enriching.
• 00:05Rare populations of primary
• 00:06human CD 34 positive cells
• 00:08by fax and has been prepared by members
• 00:10of the Kraus lab at Yale School of
• 00:13Medicine in partial fulfillment of
• 00:15the educational aims outlined in
• 00:17the mission of the Yale Cooperative
• 00:19Center for Excellence in Hematology.
• 00:21The purpose of this protocol is to analyze
• 00:25an isolate where populations of CD 34,
• 00:27positive matter, poetic stem,
• 00:29and progenitor cells from human G,
• 00:31CSF, mobilized peripheral blood,
• 00:33but maybe more broadly applicable
• 00:35to other starting cell populations.
• 00:38We will demonstrate the sorting
• 00:40strategy for human megakaryocytic
• 00:42over three progenitors in this video
• 00:44as an example of a rare population
• 00:46within the CD 34 positive fraction.
• 00:50Human CD 34 positive cells are
• 00:53a heterogeneous mixture of stem
• 00:54and progenitor cells at various
• 00:57stages of differentiation.
• 00:58To study a specific population of
• 01:01cells like high metabolic stem cells,
• 01:03this population must be enriched prior
• 01:06to any assets that may take place.
• 01:10Each progenitor population is
• 01:12defined by a combination of
• 01:14surface marker expression profiles.
• 01:17Fluorescence activated cell sorting
• 01:19or fax uses fluorescent markers to
• 01:22detect and separate the cells with
• 01:25desired surface marker expression.
• 01:27Building a customized flow cytometry panel.
• 01:31Designing a multi color panel for flow
• 01:35cytometry takes advanced preparation.
• 01:37Factors that need to be considered include.
• 01:41Configuration of the instrument.
• 01:43Such as the lasers and filters.
• 01:45It is equipped with. The number of
• 01:50colors or antibodies to be used.
• 01:53And the abundance of the antigens.
• 01:57We highly recommend utilizing Flora Finder as
• 02:01a platform for designing a multi color panel.
• 02:05Floor Finder allows the user to enter the
• 02:07exact specifications of their cytometer,
• 02:09including which lasers and filter
• 02:11sets are installed in the machine.
• 02:14Next,
• 02:14users can search and select which
• 02:17cell surface antigens and or
• 02:19fluorescent proteins they intend
• 02:21to analyze their cell sample for.
• 02:23It is useful to have prior knowledge of
• 02:26the antigen abundance on the cells in
• 02:28your sample and cell type of interest,
• 02:30but it is not necessary.
• 02:32Based on the specifications of
• 02:34the CYTOMETER and antigens to be
• 02:37detected that the user entered,
• 02:39Flora Finder populates a chart of
• 02:41commercially available antibodies
• 02:42for those antigens categorized by
• 02:44fluorescent channel such that the
• 02:47selection of 1 antibody in a particular
• 02:49channel automatically blocks the
• 02:51user from selecting additional
• 02:52antibodies in the same channel,
• 02:54thus reducing spectral overlap.
• 02:57Titrating staining antibodies.
• 02:59To ensure appropriate staining but
• 03:01not compromise antibody specificity,
• 03:03it is recommended that you titrate
• 03:06your antibodies prior to using
• 03:09them in a flow analysis or sort.
• 03:12To accomplish this utiliza cell
• 03:14sample that contains a mixture of
• 03:16cells that are positive for the
• 03:18antigen and negative pipette.
• 03:19A constant number of cells in a constant
• 03:23volume of staining solution into at
• 03:25least three but preferably 5 fax chips.
• 03:29Keep one tube as the unstained
• 03:31control and add increasing volumes
• 03:33of the antibody wish to titer to
• 03:36each subsequent tube.
• 03:37The range of volumes you choose
• 03:39should be based on the recommended
• 03:42dilution from the manufacturer.
• 03:44For example.
• 03:45If the recommended volume test for
• 03:48the manufacturer is 5 microliters,
• 03:50pipette 5 microliters of antibody
• 03:53into one tube.
• 03:55Two microliters of antibody into
• 03:57the next tube.
• 03:59One microliter of antibody into
• 04:01the next tube.
• 04:02And half a microliter of antibody
• 04:05into the last tube.
• 04:07At the Cytometer record,
• 04:09at least 10,000 events for each tube.
• 04:12Calculate the staining index by
• 04:14measuring the MFA of the negative cells
• 04:17and the standard deviation as well
• 04:19as the MFA for the positive cells.
• 04:22With these three measurements,
• 04:24calculate the staining index
• 04:26by subtracting the MFA of the
• 04:28negative population from the MFA
• 04:30of the positive population.
• 04:32And divide this by two times
• 04:34the standard deviation of
• 04:35the MFA of the negative population.
• 04:37Plot the staining index for each
• 04:39tube on a graph to identify the best
• 04:43dilution for that particular antibody.
• 04:45Sawing and staining CD. 34 positive cells.
• 04:51For the purposes of this didactic video,
• 04:54we will demonstrate the staining protocol for
• 04:57human megakaryocytic erythroid progenitors.
• 04:59You will need to substitute
• 05:00your antibodies of interest,
• 05:02but the protocol remains the same.
• 05:05With stained with CD,
• 05:0634 positive cells with a panel of
• 05:08seven antibodies, one of which is
• 05:11a cocktail of Lenny edge markers.
• 05:14To reduce the number of channels required for
• 05:16the sort and thus reduce spectral overlap,
• 05:19we use antibodies against the lineages.
• 05:21Markers that are directly
• 05:23conjugated to biotin.
• 05:25So we incorporate a secondary
• 05:26stain with streptavidin antibody
• 05:28directly conjugated to a floor.
• 05:30For that has limited spectral overlap
• 05:32with the rest of our staining panel.
• 05:37Required reagents include FBZ.
• 05:41Buffer one. Fax buffer.
• 05:45And titrated antibodies.
• 05:51Prepare labeled facts tubes
• 05:53for each compensation control.
• 05:55Fluorescence minus one control.
• 05:57As well as the sample tube.
• 06:03Compensation controls are cells
• 06:05stained with a single antibody.
• 06:08Compensation controls are required for
• 06:10every floor floor and allow the cytometry
• 06:13software to calculate and subtract
• 06:16spectral overlap between flora force.
• 06:18Fluorescence minus one controls
• 06:20are cells stained with all but one
• 06:22of the antibodies in the panel.
• 06:24It identifies the true negative of a floor
• 06:28for considering all the interference
• 06:30from the other staining floor force.
• 06:32These controls help determine the
• 06:35location of gates that separates cells not
• 06:38expressing the antigen from cells that are.
• 06:40They are required if the expression
• 06:43pattern of the antigen in your
• 06:45starting population is a continuum
• 06:48of negative to high expressing.
• 06:50Or the expression of the antigen is very low,
• 06:53so the positive signal is just
• 06:54slightly higher than the background
• 06:56fluorescence of the negative population.
• 07:00We quickly thaw cryopreserved CD
• 07:0234 positive cells in a 37 degree
• 07:05water bath until just a few ice
• 07:07crystals are left in the cryotube.
• 07:10Take care not to submerge the
• 07:12O-ring and cap of the cryo vial
• 07:14to reduce risk of contamination.
• 07:16When the cells are nearly thought,
• 07:18spray the cry of I'll with 70%
• 07:20ethanol and wipe it dry before
• 07:22bringing it into the hood to open.
• 07:25Be sure to practice strict
• 07:27aseptic technique in a bio safety
• 07:29cabinet rated for BSL 2 for the
• 07:31duration of this protocol.
• 07:41Add one milliliter of 100%
• 07:43FBS to a 50 mil conical tube.
• 07:51Add one milliliter of 100% FBS to the
• 07:54cryotube and gently transfer the full
• 07:56contents of the cryotube into the
• 07:5850 mil conical tube containing FBS.
• 08:13Add up to 50 milliliters of buffer.
• 08:16One dropwise cell suspension
• 08:17in the 50 mil conical tube.
• 08:19To delete out the DMSO in the freezing media.
• 08:25Spin at 1200 RPM 4 degrees
• 08:28Celsius for 10 minutes.
• 08:33When the spin is done,
• 08:35carefully discard the supernatant.
• 08:40Resuspend the pellet with 20
• 08:43milliliters of fax buffer.
• 08:44Take a representative 10 microliter
• 08:46aliquot for cell counting and repeat the
• 08:49spin of the conical tube at 1200 RPM,
• 08:514 degrees Celsius for 10 minutes.
• 08:55Add 10 microliters of two extra pen blue
• 08:58to the aliquot of cells and pipette to mix.
• 09:02Calculate the viable cell number
• 09:03in the sample control tubes.
• 09:05Each require 5 * 10 to the four cells.
• 09:09FMO tubes require as few as
• 09:112 * 10 to the 4th cells.
• 09:15When the second spin is complete,
• 09:17resuspend the pellet in 500
• 09:19microliters of Facs buffer an
• 09:20aliquot the necessary volume of
• 09:22cells required for control tubes.
• 09:24Add additional facts buffer to
• 09:26bring the sample tube volume back
• 09:28up to 500 microliters.
• 09:29Take care not to exceed a cell
• 09:32concentration of 20 * 10 to the six
• 09:34cells per milliliter in the sample tube.
• 09:39Aliquot the titrated amount
• 09:41of primary staining antibodies
• 09:43into the corresponding tubes.
• 09:45Be especially careful when allocating
• 09:47antibodies in the FMO tube so
• 09:49you don't accidentally add the
• 09:51antibody that should be absent.
• 09:53Additionally, it is important to
• 09:55note that the volume of antibodies
• 09:57added cannot exceed the maximum
• 09:59standing volume, which is.
• 10:00100 microliters for controls
• 10:02and one milliliter for sample.
• 10:05In other words,
• 10:06if you allocated 50 microliters of
• 10:08control cells to the control tubes,
• 10:10the volume of staining antibody
• 10:12cannot exceed 50 microliters so
• 10:14that the total staining volume
• 10:17remains 100 microliters.
• 10:18If the volume of the staining
• 10:20antibodies is less than 50 microliters,
• 10:22then add additional facts buffer to
• 10:24bring the final staining volume to
• 10:26100 microliters for control tubes and
• 10:28one milliliter for the sample tube.
• 10:33Once all cells Facs buffer an,
• 10:36antibodies are added to the staining tubes,
• 10:39incubate according to the
• 10:41manufacturer's recommendations.
• 10:42Most antibodies require incubation
• 10:44for 30 minutes on ice in the dark.
• 10:49After the primary stain is complete,
• 10:51add one milliliter of fax buffer
• 10:53to the control tubes and two
• 10:56milliliters of fax buffer to the
• 10:58sample tube and spin at 1200 RPM,
• 11:004 degrees Celsius for 10 minutes.
• 11:06When the spin is complete,
• 11:08discard the supernatant, taking
• 11:09great care not to discard the cell
• 11:11pellets at the bottom of the tubes.
• 11:22If any of the antibodies used in the
• 11:24first stain were conjugated to biotin,
• 11:26a secondary stain with
• 11:28streptavidin conjugated to a
• 11:29unique floor 4 will be required.
• 11:31Similarly to the primary stain,
• 11:33add the appropriate amount of
• 11:35streptavidin according to the
• 11:37titration to the appropriate tubes.
• 11:39You don't have to add this to single
• 11:42color control tubes that were not
• 11:44stained with a biotin conjugated
• 11:46antibody or to an FMO tube in which the
• 11:49biotin conjugated antibody was omitted.
• 11:51Bring the total staining volume
• 11:53up to 100 microliters for control
• 11:56teams or one milliliter for sample
• 11:58tubes with fax buffer.
• 12:00Incubate according to the
• 12:02manufacturer's recommendation.
• 12:04Most antibodies require incubation
• 12:05for 30 minutes on ice in the dark.
• 12:12Once secondary staining is complete,
• 12:14repeat the wash and spin steps as
• 12:16was done after the primary stain.
• 12:19Resuspend the control pellets in
• 12:21200 microliters of Facs buffer
• 12:23and 300 microliters of Facs
• 12:24buffer for the sample palette.
• 12:26Filter these cell suspensions through
• 12:28a 40 or 100 Micron filter depending
• 12:30on the expected size of yourselves
• 12:32and proceed to the cytometer.
• 12:41Prime just sorting the cells.
• 12:42Prepare collection tubes or plates
• 12:44to receive the sorted cells.
• 12:46Confirmed that Uber plate you wish
• 12:48to use fits the specification
• 12:50of the cytometer to be used.
• 12:52Typically, growth media supplemented
• 12:54with survival cytokines can be used
• 12:57to collect certain cells into and
• 12:59results in better post sort viability.
• 13:02Of special note,
• 13:03be sure to follow all safety policies
• 13:05and procedures when transporting
• 13:06yourselves to the cytometer.
• 13:08For example,
• 13:09unfixed human CD 34 positive cells
• 13:11are required to be transported
• 13:13in a secondary container that is
• 13:15labeled with a BSL two sticker.
• 13:19Once at the cytometer,
• 13:21set the color parameters that
• 13:23will be used to sort the cells.
• 13:25Create compensation tubes for each
• 13:27single color control tube you have
• 13:29and create a new specimen entitled,
• 13:31FMO and generate tubes for each FML you have.
• 13:35Also create a tube for your sample.
• 13:39Using the unstained control cells,
• 13:41adjust the voltages for each channel,
• 13:43including FC and SC,
• 13:44so that your cells are mostly clustered
• 13:47in the center of the FC SC dot plot
• 13:50and the background signal in each
• 13:52color channel is well below 2:50.
• 13:56Before recording,
• 13:56check those voltages on each of
• 13:58the single color control tubes
• 14:00as well as the sample tube.
• 14:01Adjust voltages for each channel
• 14:03so that the negative peak is below
• 14:0510 to the third and the positive
• 14:07peak is below 10 to the five.
• 14:11Once you are satisfied with the voltages,
• 14:13begin by recording single color
• 14:15controls and calculate the compensation.
• 14:17Once all have been recorded.
• 14:20Then record the FML's.
• 14:22Use the recorded data from the
• 14:24Fmo's to help draw and position
• 14:26gates based on true negatives.
• 14:30Once all of the gates of the
• 14:32sorting strategy have been drawn,
• 14:33load the sample onto the cytometer and
• 14:36set the population you wish to sort.
• 14:38Also specify what kind of
• 14:40collection container you're
• 14:41using and how many cells to sort.
• 14:44Then begin sorting your sample
• 14:45tube and collecting your enriched
• 14:47population for downstream applications.
• 15:01After the service complete, use the
• 15:03cells for your downstream application.
• 15:06If sorted cells were collected in a fax tube,
• 15:09add 2 milliliters of I MDM media
• 15:11to wash the cells in the sorted
• 15:13tube and spent at 1200 RPM,
• 15:154 degrees Celsius for 10 minutes.
• 15:21Resuspend the cell pellet in the
• 15:23appropriate growth media or freezing media.
• 15:28This concludes the protocol for
• 15:31Multicolor Facs sorting of CD 34.
• 15:34Positive amount Aquatic Center gender cells.